Figure e-1. Age-dependent changes of EEG in patients 2 and 5
Both patients (2 and 5) showed a typical pattern of hypsarrhythmia consisting of high-amplitude irregular slow waves mixed with multifocal spikes or sharp waves at 3 months of age. A brief (3–5 s) interval of diffuse flattening or suppression appeared in patient 2 at 3 months and in patient 5 at 4 months of age. This changed to a symmetric or asymmetric suppression-burst pattern consisting of intermittent high-amplitude irregular slow waves intermingled with spikes and suppression phase at 10 years in patient 2 and at 5 months of age in patient 5. The horizontal calibration mark is 1 s and the vertical one is 100 µV. m, months; y, years.
Figure e-2. Flow cytometry of granulocytes from the patients’ mother harboring a heterozygous PIGA mutation
Flow cytometry of blood granulocytes from the mothers of patients 3 and 4 (Upper panels) and patient 5 (Lower panels). Expression of various GPI-anchored proteins is indicated (mother, thick lines; normal control, dotted lines). Mean fluorescent intensities of each sample against a normal control are shown in each panel. In the mother of patients 3 and 4 (Upper panels), there was the population with significantly reduced surface expression of CD16 whereas there was no reduced population in the mother of patient 5 (Lower panels). Light shadows represent isotype controls.
Figure e-3. Expression of wild-type and mutant PIGA proteins in PIGA-deficient B lymphoblasts
Lysates of cells shown in figure 4A, C, and E were subjected to SDS-PAGE and western blotting. PIGA mutant proteins were expressed at levels similar to or higher than that of the wild-type protein. Lane 1, empty vector; lane 2, wild-type; lane 3, I206F mutant; lane 4, R412X mutant; lane 5, R412 truncated; lane 6, empty vector; lane 7, wild-type; lane 8, R77L mutant. Quantities normalized to the intensities of the GAPDH loading control, and luciferase activities for evaluating transfection efficiencies are shown below each lane.
Table e-1. Pathogenicity prediction of PIGA mutations
Case / Diagnosis / Mutation / Amino acid alteration / Inheritance / SIFT / Polyphen2 / MutationTaster / In-house database1 / Ohtahara syndrome / c.1234C>T / p.Arg412* / N. D. / N. A. / N. A. / Disease causing
1.0 / 0 / 573a
2 / Early-onset West syndrome / c.616A>T / p.Ile206Phe / Maternally
transmitted / 0.00 / Probably damaging
0.961 / Disease causing
0.999 / 0 / 573
3 and 4 / EOEE / c.230G>T / p.Arg77Leu / Maternally
transmitted / 0.00 / Probably damaging
0.996 / Disease causing
0.999 / 0 / 573
5 / West syndrome / c.355C>T / p.Arg119Trp / Maternally
transmitted / 0.00 / Probably damaging
0 / Disease causing
0.999 / 0 / 573
N. D., not determined
EOEE, early-onset epileptic encephalopathies
N. A., not applicable
SIFT (http://sift.jcvi.org/): scores of less than 0.05% indicate substitutions predicted to be intolerant
PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/): HumVar scores are evaluated as 0.000 (most probably benign) to 0.999 (most probably damaging)
Mutation Taster (http://www.mutationtaster.org/): rapid evaluation of DNA sequence alterations. The alterations are classified as disease causing or polymorphisms
a281 male and 292 female patients
Appendix e-1
Case reports
Patient 1 had a hemizygous nonsense mutation, c.1234C>T (p.R412X), which was previously reported in a family with EME.e1) This case was reported elsewhere as Schinzel-Giedion syndrome.e2) The patient was born at 33 weeks of gestation after in vitro fertilization and polyhydramnios. His parents were healthy and non-consanguineous. His birth weight was 2,857 g (+2.5 SD), his length was 42 cm (−0.6 SD), and his head circumference was 33.2 cm (+1.6 SD). He needed mechanical ventilation for respiratory distress syndrome soon after birth and received a tracheostomy at 4 months of age. He showed facial abnormalities with blepharophimosis and a depressed nasal bridge, a high-arched palate, hepatomegaly caused by hepatoblastoma, micropenis, vesicoureteral reflux, bilateral inguinal herniation, and joint contractures. He showed tonic seizures with suppression burst on EEG at 1 month and was diagnosed with Ohtahara syndrome. Soon after this, he was diagnosed with EME as the type of his seizures shifted to erratic myoclonus associated with a burst of electrical activity on EEG. Brain atrophy, a hypoplastic corpus callosum, delayed myelination, and abnormally high signals on diffusion-weighted images in the bilateral thalamus and brainstem were seen on MRI in infancy. At 6 years of age, he showed hypotonia, profound intellectual disability, and frequent myoclonus. No mutation was identified in SETBP1, which is the gene responsible for Schinzel-Giedion syndrome.
Patient 2 had a hemizygous missense mutation, c.616A>T (p.I206F), inherited from his mother. He was born at 40 weeks of gestation after an uneventful pregnancy. His birth weight was 3,566 g (+1.0 SD). He showed facial abnormalities with malar flattening, upslanting palpebral fissures, a depressed nasal bridge, a short, anteverted nose, a high-arched palate, and joint contractures. He showed myoclonus or spasm-like movements at 3 months of age. His interictal EEG showed hypsarrhythmia, but no ictal discharges. His brain MRI was normal at 6 months, but delayed myelination was seen at both 2 and 8 years of age. He was diagnosed with early-onset West syndrome with hypomyelination,e3) but no mutation was identified in the causative gene, SPTAN1.e4) At the age of 8 years, he showed tonic seizures with periodic clusters of multifocal spike-and-slow wave complexes similar to a suppression burst on EEG, profound intellectual disability, spastic quadriplegia, and bulbar palsy with gastrostomy and tracheostomy.
Patient 3 had a hemizygous missense mutation, c.230G>T (p.R77L), inherited from his mother. He was born by spontaneous delivery at 38 weeks of gestation after an uneventful pregnancy. His father suffered from depression. His birth weight was 2,715 g (−1.2 SD) and his head circumference was 32.5 cm (−0.7 SD). He showed no dysmorphism except for mildly long palpebral fissures. At 7 months of age, he developed generalized tonic seizures with an elevation of the upper extremities for 10 min during sleep. His EEG and MRI were normal, but the same type of seizures occurred four more times until the age of 12 months. He was treated with valproic acid, but four episodes of febrile convulsion occurred at the age of 1 year, and afebrile hypomotor seizures followed by generalized tonic-clonic convulsions lasting 1 min occurred once a month from the age of 2 years, then increased to once a week from the age of 2 years and 4 months. Left occipital-dominant irregular spike-and-slow wave complexes and occipital slow waves were seen on his EEG at 2 years of age. His tonic seizures with a convergence of head and eyes to the right disappeared after topiramate (TPM) medication started at the age of 2 years and 11 months. He sat without support at 7 months of age, and began to walk alone at 16 months of age. He started to speak at 18 months of age, but his meaningful words disappeared from 3 years of age and he showed a regression of his developmental quotient (DQ) score from 51 at 3 years to 17 at 4 years. He also showed athetotic movement from 3 years and 3 months of age and autistic behavior. An examination of plasma amino acids showed a temporal elevation of histidine and beta-alanine, but cerebrospinal fluid and EEG were normal. TPM medication was stopped at 4 years of age, after which he began to develop gradually. His EEG at 5 years and 7 months showed irregular spike-and-slow waves and multifocal spikes. He showed repeated attacks of cyclic vomiting from 5 to 7 years of age.
Patient 4 was a younger brother of patient 3. He was born at 36 weeks of gestation with no asphyxia. His birth weight was 1,836 g. He sat without support at 7 months of age and walked alone at 16 months. He showed generalized tonic seizures and generalized clonic seizures at 7 months, but his physical findings including his facial appearance were normal. Three episodes of febrile seizures were seen up to the age of 14 months as well as an episode of afebrile seizure demonstrating eye convergence and pale face followed by generalized clonic convulsion. At 18 months of age, he was able to run but did not respond to calling his name or simple orders, and his DQ score was 54.
Patient 5 was the first child of nonconsanguineous healthy parents. He showed polyhydramnios during pregnancy and was born at 39 weeks of gestation. His birth weight was 3,486 g (+0.7 SD), his length was 47 cm (−1.4 SD), and his head circumference was 33.5 cm (±0 SD). He needed resuscitation soon after birth because of systemic cyanosis and muscular hypotonia and was admitted to a neonatal intensive care unit. He showed facial dysmorphism such as a depressed nasal bridge, a large mouth, a high-arched palate, ankyloglossia, a submucous cleft palate, micrognathia, and other minor anomalies including a narrow anterior fontanel, a single transverse palmar crease, a prominent calcaneus, a left inguinal hernia, and a hydrocele testicle. He suffered from pneumothorax at 1 day old. His brain MRI at 1 month old showed hyperintensity on T1-weighted images at the bilateral basal ganglia and corona radiata. He needed tube feeding and home-oxygen therapy because of poor sucking and weak respiration, respectively. He showed epileptic spasms and erratic myoclonic seizures composed of repetitive episodes of thrashing of the extremities, eyelid twitching, hiccupping, and a bad temper at 3 months of age. His EEG demonstrated hypsarrhythmia and he was diagnosed with West syndrome. Multiple anti-epileptic drugs including pyridoxine, zonisamide, clobazam, valproic acid, lamotorigine, clonazepam, and adrenocorticotropic hormone injections were not effective for his seizures, and levetiracetam worsened them. At 4 months of age, TPM and lamotrigine caused the seizures to stop, and relatively longer desynchronization or suppression phases (3–5 s) appeared on the EEG. At 5 months of age, his EEG demonstrated symmetric or asymmetric suppression burst patterns. A brain MRI at 9 months of age showed progressive brain atrophy, delayed myelination, a thin corpus callosum, and restricted diffusion pattern at the brainstem and deep white matter. He suffered a relapse of epileptic spasms and myoclonic seizures at 12 months of age and they were refractory to medications.
e-References
e1) Johnston JJ, Gropman AL, Sapp JC et al: The phenotype of a germline mutation in PIGA: the gene somatically mutated in paroxysmal nocturnal hemoglobinuria. Am J Hum Genet 2012;90:295-300.
e2) Watanabe S, Murayama A, Haginoya K et al: Schinzel-Giedion syndrome: a further cause of early myoclonic encephalopathy and vacuolating myelinopathy. Brain Dev 2012;34:151-5.
e3) Tohyama J, Akasaka N, Osaka H et al: Early onset West syndrome with cerebral hypomyelination and reduced cerebral white matter. Brain Dev 2008;30:349-55.
e4) Saitsu H, Tohyama J, Kumada T et al: Dominant-negative mutations in alpha-II spectrin cause West syndrome with severe cerebral hypomyelination, spastic quadriplegia, and developmental delay. Am J Hum Genet 2010;86:881-91.