De novo mutations in GRIN1 cause extensive bilateral polymicrogyria

NMDA receptor agonists have been used for many years to generate animal models of polymicrogyria, a malformation of cortical development. Fry et al. identify de novo GRIN1 mutations in eleven patients with severe bilateral polymicrogyria. Polymicrogyria-associated GRIN1 mutations cluster in specific protein domains and significantly alter NMDA receptor function.

fissures, small upturned nose, thin upper lip, small chin and an excoriated rash around her neck (due to the dribbling). MRI at 5 months of age showed extensive polymicrogyria with occipital sparing.
The lateral ventricles were enlarged and the periventricular white matter was reduced. Corpus callosum and posterior fossa were normal. Metabolic investigation, TORCH screen, routine karyotype and array CGH were all normal. It is unknown if the TORCH screen included testing for cytomegalovirus.

Patient 3
This female patient was the child of non-consanguineous French parents. The pregnancy was complicated by maternal diabetes. The patient was born by caesarean section at 38+5 weeks gestation. She had transient respiratory distress following delivery. Her birth weight was 2.8kg (-0.9 SD), length 49cm (-0.2 SD) and OFC 34cm (0.0 SD). There were no early feeding problems reported. She presented at 2 months of age with infantile spasms. The patient was last reviewed at 4 years and 7 months of age. She had profound developmental delay. There was no history of regression. The patient was unable to sit unsupported and had no language development. She had cortical visual impairment. There were no concerns about hearing. She was gastrostomy fed and had problems with severe dribbling and intermittent constipation. Seizure types include frequent myoclonic jerks, daily spasms, and occasional tonic-clonic seizures. On examination, the patient had an OFC of 43 cm (-7.1 SD), height was 93 cm (-3.0 SD) and weight was 15.4 kg (-1.0 SD). She had spastic quadriplegia with axial hypotonia, severe dribbling and strabismus. MRI brain scan was reported to show diffuse bilateral polymicrogyria with thin corpus callosum. Basal ganglia, brainstem and cerebellum were normal. Array CGH and sequencing of GPR56 and TUBB2B were normal.

Patient 4
This male patient was the child of non-consanguineous French parents. He was born by caesarean section at 40 weeks gestation. Birth weight was 3.58kg (+0.1 SD), length 49cm (-0.2 SD) and OFC 34cm (-0.9 SD). Concerns were raised in the neonatal period because of irritability, frequent crying and episodes of hyperextension. There were no early feeding problems. Tonic seizures began at 3 months of age. The patient was last reviewed at 19 months of age. He had severe developmental delay. He was not sitting and had no language development. The patient had weekly spasms, cortical visual impairment, problems with constipation and was gastrostomy fed. On examination his OFC was 47.5cm (-1.5 SD), length 79cm (-1.3 SD) and weight 15kg (+2.4 SD). He had spastic quadriplegia, axial hypotonia and roving eye movements. He was not considered to be dysmorphic.
An MRI brain scan at 3 months of age showed extensive polymicrogyria with occipital sparing.
Extensive metabolic investigation was normal.

Patient 5
This male patient was the child of non-consanguineous Israeli parents. He was born by normal vaginal delivery at 40+2 weeks gestation following an uncomplicated pregnancy. Birth weight was 3.37kg (-0.4 SD). His head size was noted to be small at birth (OFC 32cm, -2.5 SD). In the first week of life he developed episodes of facial grimacing, screaming and hand fisting. These were considered to be seizures. Drug therapy (phenobarbital followed by vigabatrin, topiramate, clobazam and lamotrigine) was initially ineffective. Ketogenic diet and steroid treatment helped reduce seizure frequency. The patient was last reviewed at 3 years and 6 months of age. He had profound developmental delay with no motor or speech development. He had severe cortical visual impairment (no fixing and following, blinking only in a dark room when a direct light beam was close to his eyes). In contrast he responded promptly, usually by smiling, to noise or tactile stimuli.
He was fed pureed food orally. The patient had been seizure free for around 18 months on a mixture of clobazam, levetiracetam and prednisolone. On examination he had severe microcephaly (OFC 42.5cm, -6.5 SD), spastic quadriplegia, axial hypotonia and roving eye movements. Height was 87cm (-3.1 SD) and weight 13.5kg (-1.3 SD). MRI brain scan at 6 weeks of age showed extensive bilateral polymicrogyria affecting the frontal, parietal, and temporal lobes. There was delayed myelination and mildly enlarged lateral ventricles. Underdevelopment of the hippocampi was noted.
EEG initially showed left temporal spike activity with subsequent bilateral multifocal epileptic discharges. Metabolic investigation, cytomegalovirus testing and array CGH were all normal.

Patient 6
This female patient was the child of non-consanguineous Israeli parents. There was no family history of learning disability, epilepsy or polymicrogyria. She was born at term by normal vaginal delivery following an uncomplicated pregnancy. Birth OFC was 33.5cm (-0.8 SD). The patient presented in the first year of life with infantile spasms. The patient was last reviewed at 9 years of age. She had severe developmental delay and was being educated in a special needs school. The patient could sit unsupported and walk with a walking frame. She had no speech but could communication through a tablet computer using her left arm. The patient had some behavioural problems with severe tantrums and autistic features. It was not clear if she smiled socially. Seizures were controlled with sodium valproate. She had oromotor problems but was still orally fed. On examination the patient had spastic quadriplegia, strabismus, pseudobulbar palsy and stereotypic movements. MRI brain scan at 8 months of age showed bilateral perisylvian polymicrogyria extending into the posterior frontal, anterior parietal and temporal lobes. Array CGH was normal.
Urine PCR and serum IgM were positive for cytomegalovirus at age 4 months, but her mother was IgG and IgM negative at 36 weeks.

Patient 7
Fetal ultrasound found this single live male fetus had moderate ventriculomegaly (15 mm), abnormal thinning and sulcation of the cerebral cortex, dysplasic corpus callosum, small head size (5th percentile, -1.6 SD) and shortened long bones (2nd-5th percentile). The fetus had clenched hands, left clubbed foot and a right rocker bottom foot. The fetus was 22 weeks gestation by dates but only 20 weeks and 6 days based by size. Fetal brain MRI showed severe parenchymal thinning throughout both cerebral hemispheres, with moderate to severe ventriculomegaly. There was abnormal cortical infolding along the left frontal lobe. There was a hypoplastic corpus callosum and mildly enlarged cerebellum. The brainstem appeared normal. The pregnancy was terminated. The parents declined fetal autopsy. The parents were non-consanguineous and had a mixed European heritage. There was no family history of brain malformations. The fetus had a normal array CGH.

Patient 8
This female patient was the child of non-consanguineous parents of mixed European heritage.
There was no family history of learning disability, epilepsy or polymicrogyria. Clubfeet were noted on prenatal ultrasound at 22 weeks gestation. She was born at 32+6 weeks gestation by caesarean section. Birth weight was 624 g (-4.7 SD), length 36 (-4.9 SD) and OFC 31 cm (+0.59 SD).
Seizures began in the neonatal period with gaze deviation, tonic stiffening and desaturations. Severe developmental delay was noted in the first months of life. The patient was last reviewed at 20 months of age. She was not sitting yet and had no language development. The patient was gastrostomy fed. Her problems included cortical visual impairment, bilateral club feet, constipation, poor linear growth with excessive weight gain, chronic restrictive lung disease, secundum atrial septal defect and spina bifida occulta. The patient continued to have daily seizures associated with gaze deviation and tonic stiffening. On examination the patient had severe microcephaly (OFC 39cm [at 1 year 10 months], -5.7 SD), spastic quadriplegia, axial hypotonia and stereotypic movements. Length and weight (at 2 years 6 months) were 74 cm (-4 SD) and 13.81 kg (+0.53 SD) respectively. She had bitemporal narrowing, prominent midface, thick eyebrows, button nose, thick lips, and a high-arched palate. MRI brain scan at 3 months of age showed bilateral fronto-parietal polymicrogyria, diffuse cerebral volume loss, dilated lateral and third ventricles, enlarged extra-axial spaces, diffuse abnormal white matter signal (likely secondary to delayed myelination), thin corpus callosum and abnormal hippocampi bilaterally with incomplete folding. Metabolic investigation and array CGH were both normal.

Patient 9
This male patient was the second child of non-consanguineous African American parents. There was a family history of epilepsy but not learning disability or polymicrogyria. He had one older and one younger brother who were healthy. He was born by normal vaginal delivery at 39 weeks gestation. He had frequent vomiting during the first weeks of life. His feeding was slow and associated with tongue biting and frequent aspiration. Seizures began at 5 weeks. He had tonic seizures (bunched up body, clenched fists and red in face), possibly infantile spasms, which were initially occurred individually but later clustered. Subsequently he had intractable seizures with myoclonic jerks and generalised tonic-clonic seizures (sometimes 30+/day). Medication tried included sodium valproate, prednisolone, lamotrigine and lorazepam. At the age of 9 he was given vigabatrin which seemed to help the best. The patient had severe developmental delay. He had a gastrostomy placed at age 8 years. On examination he had a mild scoliosis. He was not dysmorphic.
The patient had severe respiratory problems with recurrent aspiration and pneumonias. He died at the age of 14 years. MRI brain scan at 4 months of age showed large extra-axial spaces with extensive frontal, perisylvian, parietal and temporal polymicrogyria. The posterior lateral ventricles were dysplastic and moderately enlarged. Corpus callosum, cerebellum and brain stem were normal. Array CGH was normal.

Patient 10
This male patient was the fifth child of non-consanguineous parents of mixed European heritage.
There was a family history of epilepsy but not learning disability or polymicrogyria. He had five siblings (4 older, 1 younger) who were all well. The pregnancy was complicated by maternal anaemia and a urinary tract infection for which his mother took an antibiotic. He was born by normal vaginal delivery at 36 weeks gestation. Birth weight was 2.64 kg (-0.1 SD), length 52.1 cm (+2.3 SD) and OFC 26.4 cm (-4.9 SD). There were concerns about his head size and possible seizures in the neonatal period. At 3 months of age he was noted to have unusual eye opening/closing and hardly ever opened his eyes. He smiled socially around 10 months. At 16 months of age his length was 69 cm (-4.0 SD), weight 8.7 kg (-2.3 SD) and OFC 40.5 cm (-6.7 SD).
The patient was diagnosed with seizures at 3 years of age. Initially these took the form of spells of abnormal gaze (he would look upward and get stuck there) with little or no responsiveness during spells. The seizures were well controlled on medication (zonisamide). He continued to have occasional gelastic seizures during illnesses and other stressors. At 5 years of age he began having recurrent aspiration, vomiting and poor weight gain. He had a gastrostomy soon afterwards. The patient had severe developmental delay. By the age of 17 years he was not sitting unsupported yet.
He had nystagmus, esotropia, and little visual tracking. His hearing was good and he made some vocalisations. On examination he had severe scoliosis, contractures of the hips, small penis, and truncal hypotonia with spastic quadriparesis. MRI brain scan at 8 months revealed diffuse bilateral polymicrogyria, mildly-enlarged extra axial spaces, reduced white matter and enlarged lateral ventricles. Basal ganglia, corpus callosum, cerebellum and brain stem were normal. Array CGH was normal.

Patient 11
This female patient was the only child of non-consanguineous White American parents. There was a family history of epilepsy but not learning disability or polymicrogyria. The pregnancy was complicated by hyperemesis and breech presentation. She was born by caesarean section at 39 weeks gestation. She fed poorly and had severe frequent vomiting during her first 6 months. She had a gastrostomy at 6 months of age. Fundoplication was performed at 1 year. Post-operative complications meant that she was unable to get enough nutrition without vomiting or retching. A gastro-jejunal tube was tried but would not stay in. She had a Roux-en-Y jejunostomy at age 7 years. Seizures began at 2 weeks of age. Initial these included tonic episodes (stiff, apnoeic and blue) and generalized tonic clonic seizures. The seizures were continuous in the first year of life. At 2-3 years of age she was still having frequent daily seizures (10-50/day). The seizures were individually short, lasting less than 1 minute, but often occurred back to back in clusters lasting up to 5 minutes. The seizures were characterised by stiffness and staring but little jerking apart from a quick jump or startle at the start. At ages 5-7 years the 1-5 minute spells continued but she was also having longer more severe seizures associated with prolonged stiffness and apnoea, followed by clonic jerking, jaw clenching and teeth grinding. She also had prolonged post-ictal sleepiness (up to 24 hours). Persistent limb spasticity was noted at 18 months of age. She had a lot of back arching from early infancy although this was thought to be due to reflux. Her OFC at 3 years and 3 months was 42 cm (-7.1 SD). The patient had severe global developmental delay. She was able to smile socially but unable to sit unsupported. She had abnormal visual orienting behaviour possibly due to delayed visual maturation superimposed on cortical visual impairment. She had a neurogenic bladder. The patient had markedly increased seizures in the last year of life and died at the age of 8 years. MRI brain scan at 2 months showed marked microcephaly with diffuse bilateral polymicrogyria. There were mildly enlarged extra axial spaces, mildly thin leaves of the hippocampus, reduced white matter volume, enlarged lateral ventricles and thin corpus callosum.
Basal ganglia, cerebellum and brain stem were normal. Repeat MRI brain at 3 years showed increased brain atrophy.

Exome Sequencing
For Patients 1 and 2, genomic DNA was extracted from blood and sequenced using a Nextera Rapid Capture Expanded Exome kit (Illumina) followed by 100 bp paired-end sequencing on an Illumina HiSeq2000. Reads were mapped to the 1000 Genomes version of the human reference genome (human_g1k_v37) using the Burrows-Wheeler Aligner v0.7.5a (Li and Durbin, 2009). Singlenucleotide variants and indels were jointly called and genotyped using the Genome Analysis Tool Kit (GATK v3.3.0) (McKenna et al., 2010) and annotated using SnpEff v3.3 and SnpSift (Cingolani et al., 2012a;Cingolani et al., 2012b). Variants were annotated for allele frequency using ExAC data (release 0.3, 14th January 2016) (Lek et al., 2016). De novo mutations were identified using the GATK SelectVariants walker according to previously described criteria (Epi4K Consortium et al., 2013). We excluded synonymous variants and variants with minor allele frequency >0.001. All putative de novo mutations were validated by Sanger sequencing in the proband and both parents.
For Patients 3 and 4, library generation, enrichment, whole exome sequencing and variant calling were performed as previously described (Poirier et al., 2013). Additional de novo mutations were found in subjects 1-4 (Supplementary Table 5). These were considered unlikely to explain the patients' phenotypes.
After 24 hours, cells were washed with Hank's Balanced Salt Solution (HBSS) supplemented with 10 mM HEPES, then the surface activity was measured by adding 100 µL of a 100 µM nitrocefin (Millipore, Billerica, MA) solution in HBSS with HEPES to the wells. To determine total activity, the cells were lysed in 50 µl H 2 O for 30 min prior to the addition of 50 µl of 200 µM nitrocefin and the absorbance was read at 468 nm every min for 30 min using a microplate reader at 30°C. All reagents were purchased from Sigma (unless otherwise stated). Data were expressed as mean standard error of the mean. Significance for all tests using unpaired t-test was set at P < 0.05.