The phenotype of a germline mutation in PIGA: the gene somatically mutated in paroxysmal nocturnal hemoglobinuria

Abstract

Phosphatidylinositol glycan class A (PIGA) is involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. Many proteins, including CD55 and CD59, are anchored to the cell by GPI. Loss of CD55 and CD59 on erythrocytes causes complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), a disease that manifests after clonal expansion of hematopoietic cells with somatic PIGA mutations. Although somatic PIGA mutations have been identified in many PNH patients, it has been proposed that germline mutations are lethal. We report a family with an X-linked lethal disorder involving cleft palate, neonatal seizures, contractures, central nervous system (CNS) structural malformations, and other anomalies. An X chromosome exome next-generation sequencing screen identified a single nonsense PIGA mutation, c.1234C>T, which predicts p.Arg412(∗). This variant segregated with disease and carrier status in the family, is similar to mutations known to cause PNH as a result of PIGA dysfunction, and was absent in 409 controls. PIGA-null mutations are thought to be embryonic lethal, suggesting that p.Arg412(∗) PIGA has residual function. Transfection of a mutant p.Arg412(∗) PIGA construct into PIGA-null cells showed partial restoration of GPI-anchored proteins. The genetic data show that the c.1234C>T (p.Arg412(∗)) mutation is present in an affected child, is linked to the affected chromosome in this family, is rare in the population, and results in reduced, but not absent, biosynthesis of GPI anchors. We conclude that c.1234C>T in PIGA results in the lethal X-linked phenotype recognized in the reported family.

Figure 1

Phenotype of IV-2 and IV-4 (A–C) Individual IV-2. (A) Craniofacial features including micrognathia, a prominent occiput, a depressed nasal bridge, a short, anteverted nose, malar flattening, upslanted palpebral fissures, a small, triangular mouth, downturned corners of the mouth, and a short neck. (B) Sagittal T1-weighted cranial MRI demonstrates thinning of the genu of the corpus callosum, mild sulcal prominence, and cerebellar hypoplasia. (C) Axial T1-weighted MRI demonstrates white-matter or myelination immaturity for the individual's age. (D–F) Individual IV-4. (D) Facial image showing similar facial features to those of IV-2. (E) Sagittal T1 cranial MRI demonstrates a thin corpus callosum, sulcal prominence, and a small cerebellum. (F) Axial T1-weighted cranial MRI demonstrates white-matter immaturity.

Figure 2

Identification and Segregation of the PIGA Mutation (A) Pedigree showing segregation of carrier or affected status with the presence of the mutation (+, wild-type; M, mutant). (B) Filtering criteria used for identification of the mutation from a total of 1,360 variants identified in individual III-2. (C) Sanger sequence verification; an arrow shows the position of the mutation.

Figure 3

Reduced Activity of the p.Arg412^∗ PIGA Mutant in Restoring Surface Expression of GPI-Anchored Protein after Transfection into PIGA-Null Cell Lines Flow-cytometry analysis showing that cell-surface expression of CD59, a GPI-anchored protein, is absent in both LD cells (A, left panel) and TF1-PNH cells (A, right panel). Seventy-two hours after transfection, the cells that received vector expressing wild-type PIGA (pPB-wtPIGA) significantly upregulated surface CD59 expression (C) in comparison to those that received empty vector controls (B). The expression of mutant PIGA (pPB-mutPIGA) partially restored the surface expression of CD59 (D).