Glycosylphosphatidylinositol Biosynthesis Defect Due To Novel Biallelic Pathogenic Variants in PIGW

Inherited glycosylphosphatidylinositol (GPI) deficiencies are a heterogeneous group of inherited disorders of glycosylation, caused by mutations in genes involved in GPI-anchored proteins (GPI-AP) biosynthesis. PIGW is a gene known to be involved in the early steps of the GPI-anchor biosynthesis, as well as functional studies for most patients. Biallelic mutations in PIGW have been previously linked to hyperphosphatasia with mental retardation syndrome 5, also known as glycosylphosphatidylinositol biosynthesis defect 11 (GPIBD11).

We report seven individuals, including two fetuses from six unrelated families. Whole exome sequencing and chromosome analysis were performed, with variant interpretation based on ACMG and AMP guidelines. Magnetic resonance imaging (MRI) was also conducted on some of the patients. Blood samples were collected from patients to analyze GPI-AP expression using flow cytometry on markers like CD16, CD24, and FLAER. Functional analyses were performed using PIGW KO HEK 293 cells generated with CRISPR/Cas9 technology. The cells were transfected with rat Pigw cDNA that contained patient variants. The restoration of GPI-AP expression was measured by flow cytometry. Western blotting was used to assess protein expression.

Affected patients exhibited a wide range of clinical features. Some patients presented classic GPIBD11 symptoms like developmental delay, hyperphosphatasia, and intellectual disability. Other patients showed atypical or milder phenotypes. The magnetic resonance imaging scans revealed variable neurological abnormalities in the affected individuals. Whole exome sequencing results identified PIGW mutations in all patients, which confirms the genetic basis of the disorder. Flow cytometry analysis of blood samples from patients P1, P4, and P5 using various markers showed a significant reduction in GPI-AP expression. The CD16 marker decreased to 1.8% in P1 and 21% in P5 compared to controls. CD24 was reduced to 22% in P1 granulocytes. Also, a minor decrease in CD14 on monocytes was observed in P4, as well as a slight reduction in the expression of FLAER in lymphocytes. Functional studies on PIGW-deficient CHO cells and HEK293 cells, using flow cytometry, showed that GPI-AP expression is affected by the PIGW variants. Western blotting showed reduced PIGW protein expression, except for P153L and R36G, which were similar to wild-type levels.

To date, six patients and two fetuses with biallelic variants in PIGW have been reported. Here, we describe five new patients and two fetuses harboring homozygous or compound heterozygous variants in the PIGW gene. Our results illustrate the clinical variability of GPIBD11, highlighting the importance of broad genomic sequencing assays for patients who do not show typical symptoms. Therefore, our study expands the clinical and molecular spectrum of PIGW-associated disorder.