A novel germline PIGA mutation in Ferro-Cerebro-Cutaneous syndrome: a neurodegenerative X-linked epileptic encephalopathy with systemic iron-overload

Abstract

Three related males presented with a newly recognized x-linked syndrome associated with neurodegeneration, cutaneous abnormalities, and systemic iron overload. Linkage studies demonstrated that they shared a haplotype on Xp21.3-Xp22.2 and exome sequencing was used to identify candidate variants. Of the segregating variants, only a PIGA mutation segregated with disease in the family. The c.328_330delCCT PIGA variant predicts, p.Leu110del (or c.1030_1032delCTT, p.Leu344del depending on the reference sequence). The unaffected great-grandfather shared his X allele with the proband but he did not have the PIGA mutation, indicating that the mutation arose de novo in his daughter. A single family with a germline PIGA mutation has been reported; affected males had a phenotype characterized by multiple congenital anomalies and severe neurologic impairment resulting in infantile lethality. In contrast, affected boys in the family described here were born without anomalies and were neurologically normal prior to onset of seizures after 6 months of age, with two surviving to the second decade. PIGA encodes an enzyme in the GPI anchor biosynthesis pathway. An affected individual in the family studied here was deficient in GPI anchor proteins on granulocytes but not erythrocytes. In conclusion, the PIGA mutation in this family likely causes a reduction in GPI anchor protein cell surface expression in various cell types, resulting in the observed pleiotropic phenotype involving central nervous system, skin, and iron metabolism.

Keywords: Ferro-Cerebro-Cutaneous syndrome; PIG-A protein; PIGA; X-linked; cerebellar atrophy; encephalopathy; epilepsy; exome; hemochromatosis; iron; microcephaly; neurodegeneration; recessive; seizures; sequencing.

FIG. 1

X chromosome alleles and pedigree. A: X chromosome is shown at the left, with the linkage area bracketed in red. The five rare variants followed by Sanger sequencing throughout the family are shown with the gene name and the location within the linkage region: PTCHD1 (g.23410745C>G), PDHA1 (g.19367346C>T), ZRSR2 (g.15838411C>T), PIGA (g. 15342943-15342945delAAG), and PRPS2 (g.12817576C>A). The PIGA gene (starred) is in the reverse orientation, so the wild type “CTT” codon for Leucine is deleted (DEL). The wild type allele (black) and the allele with the PIGA codon deletion (red) are shown, along with three other allele variations (orange, blue, and purple). B: Family pedigree. Squares are male and circles are female. Filled squares indicate affected males, including the propositus IV-3 and his affected uncle III-9 . The half-filled circles are carrier females or obligate carriers. Exome-sequenced individuals are circled.

FIG. 2

Sanger verification and multiple protein sequence alignment. A: All family members with available DNA samples were Sanger sequenced for the PIGA three base pair deletion variant position (marked with a bar, CTT). The example sequences shown are in the reverse direction, so the carriers (blue text label) have two sequences to the left of the mutation position due to being heterozygous for the three base pair deletion. The affected individuals have a red text label. The other family members not shown were all wild type at this position. B: Multiple protein sequence alignment over the deleted amino acid of the PIGA gene (p.Leu110, boxed in red) is shown from human to fish and fruit fly sequences.

FIG. 3

Electron microscopic leukocyte studies demonstrated lymphocytes with small myelin figures and clusters of vacuoles with electron dense tubules; a siderosome is evident in 6a as indicated by arrowhead.

FIG. 4

Electron micrographs of brain from a frontal lobe biopsy in III-9 at age 5 years demonstrated abnormal appearing mitochondria and fibrillary astrocytic processes.

FIG. 5

Photos of affected males. (a) Individual III-10 at age 6 months, and (b) at age 5 years. (c) Individual IV-3 at 6 months and (d) vegetative, at age 14 years.

FIG. 6

Histological sections from kidney, liver, and spleen from autopsy (III-9). a: Brown pigments in renal tubules (H&E×100). b: The pigments were positive for iron-Prussian blue stain (×100). c: Cirrhotic nodules with fatty change (H&E, ×200). d: Marked accumulation of hemosiderin in hepatocytes was present (Iron-Prussian blue stain, × 200). e: No grossly evident pigment deposition in spleen (H&E, × 100). f: Patchy iron deposition in spleen (Iron-Prussian blue stain, × 100).

FIG. 7

Scant iron deposition in brain from autopsy (III-9)—Basal Ganglia. a: Rare sections of basal ganglia showed brown pigments in neurons (H&E × 400). b: Iron-Prussian blue stain demonstrated that the pigments were positive for iron staining (×400).

FIG. 8

Evolution of skin lesions with age, individual IV-3. a: Ichthyosis, age 5½ years. b: Yellowish brown plaque-like neck lesions, age 14 years. c: Streaky axillary and trunk hyperpigmentation, age 5½ years. d: Elbow lesions, age 14 years.

FIG. 9

Cutaneous and oral abnormalities, individual IV-3. (a) Epidermal microabscesses (b) microdontia, with widely spaced pointed teeth and gingival overgrowth are representative of those in all three affected males. c: Foot deformity and skin lesions, age 14 years.

FIG. 10

Neuroimaging features. Serial MRI images in individual IV-3 demonstrated progressive cerebellar and to a lesser extent cerebral atrophy and white matter loss at ages (a) 8 months, (b) 3½ years, and (c) 5½ years. d: Stir images performed at age 5½ years failed to demonstrate excess brain iron deposition.

FIG. 11

Experiments showing GPI-anchored protein levels in blood cells. A and B: Cell surface expression of GPI-anchored proteins was assayed by flow cytometry. FLAER is a stain for GPI expression. The sample from IV-3 is the black square, and the 10 normal controls are the gray circles. A: FLAER and CD24 were tested for the granulocytes. B: FLAER and CD14 were tested for the monocytes. C: CD59 cell surface expression on the red blood cells. The gray bar is the average for ten controls (with SD marked), and the black bar is IV-3’s data.