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Case Reports
. 2023 Jan 30;14(2):359.
doi: 10.3390/genes14020359.

Excluding Digenic Inheritance of PGAP2 and PGAP3 Variants in Mabry Syndrome (OMIM 239300) Patient: Phenotypic Spectrum Associated with PGAP2 Gene Variants in Hyperphosphatasia with Mental Retardation Syndrome-3 (HPMRS3)

Miles D Thompson  1 , Xueying Li  2 , Michele Spencer-Manzon  3 , Danielle M Andrade  1   4 , Yoshiko Murakami  2 , Taroh Kinoshita  2 , Thomas O Carpenter  5
Affiliations
Case Reports

Excluding Digenic Inheritance of PGAP2 and PGAP3 Variants in Mabry Syndrome (OMIM 239300) Patient: Phenotypic Spectrum Associated with PGAP2 Gene Variants in Hyperphosphatasia with Mental Retardation Syndrome-3 (HPMRS3)

Miles D Thompson et al. Genes (Basel). .

Abstract

We present a case report of a child with features of hyperphosphatasia with neurologic deficit (HPMRS) or Mabry syndrome (MIM 239300) with variants of unknown significance in two post-GPI attachments to proteins genes, PGAP2 and PGAP3, that underlie HPMRS 3 and 4.

Background: In addition to HPMRS 3 and 4, disruption of four phosphatidylinositol glycan (PIG) biosynthesis genes, PIGV, PIGO, PIGW and PIGY, result in HPMRS 1, 2, 5 and 6, respectively.

Methods: Targeted exome panel sequencing identified homozygous variants of unknown significance (VUS) in PGAP2 c:284A>G and PGAP3 c:259G>A. To assay the pathogenicity of these variants, we conducted a rescue assay in PGAP2 and PGAP3 deficient CHO cell lines.

Results: Using a strong (pME) promoter, the PGAP2 variant did not rescue activity in CHO cells and the protein was not detected. Flow cytometric analysis showed that CD59 and CD55 expression on the PGAP2 deficient cell line was not restored by variant PGAP2. By contrast, activity of the PGAP3 variant was similar to wild-type.

Conclusions: For this patient with Mabry syndrome, the phenotype is likely to be predominantly HPMRS3: resulting from autosomal recessive inheritance of NM_001256240.2 PGAP2 c:284A>G, p.Tyr95Cys. We discuss strategies for establishing evidence for putative digenic inheritance in GPI deficiency disorders.

Keywords: CD55; CD59; Mabry syndrome; developmental disability; exome panel sequencing; flow cytometry; glycosylphosphatidylinositol (GPI) disorder; hyperphosphatasia with mental retardation (HPMRS); post attachment to proteins; rescue assay; type 2 (PGAP2).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The effect of c.284A>G mutant hPGAP2 on the expression of GPI-anchored proteins (GPI-APs) in PGAP2 deficient CHO cells. Isotype (grey); PME WT FLAG-hPGAP2 (red); PME c.284A>G mutant FLAG-hPGAP2 (blue); PME empty (yellow). Compared with wide type hPGAP2, hPGAP2 c.284A>G could not rescue expression of GPI-APs, (A). CD59; (B). CD55; (C). uPAR; (D). FLAER in PGAP2 deficient CHO cells. (E). Western blot analysis of the FLAG hPGAP2 proteins in CHO cells. The result showed that hPGAP2 mutant protein (p.Try95Cys) was not detected, while the wild-type hPGAP2 protein was detected at 26 kDa by the anti-FLAG antibody. The mutation destabilized the PGAP2 protein expression.
Figure 2
Figure 2
The effect of c.259G>A mutant hPGAP3, driven by the strong pME promoter, on the expression of GPI-APs in PGAP2- and PGAP3-deficient Chinese hamster ovary (CHO) cells. Isotype (grey); PME WT hPGAP3-HA (red); PME c.284A>G mutant hPGAP3-HA (blue); PME empty (yellow). The wide type hPGAP3 and hPGAP3 c.259G>A reduced the expression of GPI-APs to similar extents (A). CD59; (B). CD55; (C). uPAR; (D). FLAER in PGAP2- and PGAP3- double deficient CHO cells.
Figure 3
Figure 3
The effect of c.259G>A mutant hPGAP3, driven by the pCMV promoter, on the expression of GPI-APs in PGAP2- and PGAP3-deficient Chinese hamster ovary (CHO) cells. Isotype (grey); pCMV WT hPGAP3-HA (red); pCMV c.284A>G (p. Val87Met) mutant hPGAP3-HA (blue); pCMV empty (yellow). The wide type hPGAP3 and hPGAP3 c.259G>A reduced the expression of GPI-APs to similar extents (A). CD59; (B). CD55; (C). uPAR; (D). FLAER in PGAP2- and PGAP3- double deficient CHO cells. (E). Western blot analysis of the hPGAP3-HA proteins expressed in CHO cells by the pCMV promoter. The glycosylated PGAP3 proteins were detected by the anti-HA antibody. As for the quantity, each band intensity was normalized by that of GAPDH as a loading control and by luciferase activities as a transfection efficiency.
Figure 4
Figure 4
The likely pathogenic PGAP2 variant is shown in context of the isoform 8 (arrow). PGAP2 c.284A>G in the transcript 12, isoform 8 (NM_001256240) encoding p.Try95Cys was tested in the rescue assay. Transcript 1 encodes the less active isoform 1 whereas transcript 12, lacking 61 amino acids translated from exon 3, encodes the more active form as previously described [28]. The predicted topology of the PGAP2 protein was modeled using TMHMM (http://cbs.dtu.dk/services/TMHMM/) (accessed on 1 December 2022).
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References

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