Clinical phenotype
Patients II-1 and II-2, dizygotic male twins and first children of non-consanguineous Caucasian parents with an unremarkable family history were born in the 30th week of gestation due to maternal HELLP syndrome (Fig. 1). Birth measures were normal for gestational age (II-1: weight 1555 g, length 43 cm; II-2: weight 1410 g, length 41 cm) and no congenital anomalies were present. Both children developed respiratory distress syndrome and were treated with surfactant. At age 3 months recurrent prolonged apnea occurred necessitating stimulation mainly during sleep. Multiple EEGs did not show epileptic activity during these episodes that persisted until age 2 years when they were last seen, despite anticonvulsive therapy. Psychomotor development was severely delayed in both children. At a corrected age of 12 months they showed pendular eye movements with rotation nystagmus, no fixation or tracking. Generalized muscular hypotonia was present with poor head control and paucity of spontaneous movements. Increased tendon reflexes with positive Babinski sign were noted as well as frequent myoclonus and tremor. At age 25 months II-1 was able to roll over and momentarily sit unsupported. He was very alert and friendly, fixated and played with objects. He could babble but did not speak. He showed frequent myoclonus and a pronounced hyperexcitability. Patient II-2 developed epilepsy during the 2nd year of life with atone seizures, desaturation, gaze deviation and smacking movements that were successfully treated with topiramate. At age 25 months he showed less developmental progress than his brother. He was able to roll on the side and grasp objects but did not interact with the caretakers. Like his brother he showed myoclonus and was hyperexcitable. At age 2 years both children presented with normal height and length. Head circumference was on the 3rd centile in patient II-1 and below the 3rd centile in patient II-2.
Investigations for metabolic disorders including serum levels for alkaline phosphatase, calcium, parathyroid hormone and vitamin D, that have been found abnormal in patients with other defects in the synthesis of GPI-anchors, were repeatedly normal [8]. Nerve conduction velocity as well as acoustic and visual evoked potentials measured in patient II-2 were within the normal range. Cerebral MRI at age one year showed frontal accentuated brain atrophy and significantly delayed myelination in T2-weighted images. On follow-up MRI at age two years the amount of myelin was increased, however the magnetization transfer (MT) saturation maps still revealed a striking reduction of signal arising from myelin compared to age matched controls. Brain atrophy was also less pronounced in the follow up MRI (Fig. 2). MR spectroscopy at age two years demonstrated a significant reduction (>2 SD of mean control values [9]) of creatine and choline concentration in both patients which may point to a decreased amount of choline containing myelin proteins and cell density a finding that has been described in hypomyelinating conditions [10] (Fig. 3).
Molecular Analysis
The exome analyses yielded 65.96 million confidently mapped reads with a mean coverage of X69. Following alignment to the reference genome, 21,931 on-target variants were noted. We removed variants which were called less than X8, were off-target, synonymous (>4 bp from exon/intron junction), MAF > 0.1 % at dbSNP138 and MAF > 1 % in the Hadassah in-house dbSNP. 98 variants survived this filtering process but only a single couple resided in the same gene, PGAP1. These were chr2: 197781284InsT, NM_024989:c.334_335insA:p.A112fs and Chr2: 197755552 C > G, NM_024989:c.G1173C:p.L391L, affecting the donor splice site of exon 10.
Sanger sequencing showed that both patients were compound heterozygous for the insertion, c.334_335InsA (p.A112fs) which was shown to be inherited from the mother, leading to a frame shift and the splice site mutation, c.G1173C (p.L391L), in the PGAP1 gene which was shown to be inherited from the father. cDNA analysis from whole blood of patients and a healthy control revealed on the one hand a transcript variant without exon 9 in patients as well as in healthy control. Additionally the splice site mutation c.G1173C (p.L391L) leads to skipping of exon 10 (Fig. 4). Thereby it is to suppose that the skipping of the exon 10 leads to a truncated protein.
For detailed information about WES and Sanger sequencing see Additional file 1.