ABSTRACT We report on two consanguineous sibs affected with severe intellectual disability and autistic features due to a homozygous missense variant of _GRIN1_. Massive parallel sequencing

was performed using a gene panel including 450 genes related to intellectual disability and autism spectrum disorders. We found a homozygous missense variation of _GRIN1_ (c.679G>C;

p.(Asp227His)) in the two affected sibs, which was inherited from both unaffected heterozygous parents. Heterozygous variants of _GRIN1_, encoding the GluN1 subunit of the NMDA receptor,

have been reported in patients with neurodevelopmental disorders including epileptic encephalopathy, severe intellectual disability, and movement disorders. The p.(Asp227His) variant is

spectrum of _GRIN1_ variants and support the existence of hypomorphic variants causing severe neurodevelopmental impairment with autosomal recessive inheritance. SIMILAR CONTENT BEING VIEWED

BY OTHERS NOVEL DE NOVO PATHOGENIC VARIANT IN THE _GNAI1_ GENE AS A CAUSE OF SEVERE DISORDERS OF INTELLECTUAL DEVELOPMENT Article 25 November 2021 A HOMOZYGOUS VARIANT IN ARHGAP39 IS

ASSOCIATED WITH LETHAL CEREBELLAR VERMIS HYPOPLASIA IN A CONSANGUINEOUS SAUDI FAMILY Article Open access 25 October 2024 SHQ1-ASSOCIATED NEURODEVELOPMENTAL DISORDER: REPORT OF THE FIRST

HOMOZYGOUS VARIANT IN UNRELATED PATIENTS AND REVIEW OF THE LITERATURE Article Open access 22 February 2023 INTRODUCTION _GRIN1_ (MIM *138249) encodes the GluN1 subunit of

N-methyl-d-aspartate receptors, members of the glutamate receptor channel superfamily, which are heteromeric protein complexes with multiple subunits arranged to form a ligand-gated ion

channel. These subunits have a key role in the plasticity of synapses, which underlies memory and learning.1 _De novo_ heterozygous variants of _GRIN1_ were first identified by targeted

Sanger sequencing in two patients with intellectual disability (ID) with or without epilepsy (MIM #614254).2 Recent advances in next-generation sequencing have shown that _GRIN1 de novo_

heterozygous variants represent a recurrent cause of epileptic encephalopathy with early onset.3, 4 A recent paper provided a better delineation of the _GRIN1_ phenotypic spectrum that

includes severe ID with absent speech, muscular hypotonia, hyperkinetic movements, oculogyric crises, hand stereotypies, cortical blindness, and epilepsy.5 This article also reported for the

first time two homozygous _GRIN1_ variants in families with severe neurodevelopmental phenotypes. In the present paper we report a second family including two affected sibs with a

homozygous missense variant, thus expanding the phenotype–genotype correlations related to _GRIN1_ variants. PATIENTS AND METHODS PATIENTS The patients were born to first-cousin parents from

Morocco (Figure 1a). Parents provided informed signed consent for genetic studies on blood samples from them and their children. GENE PANEL Massive parallel sequencing was performed in

patient 1 with a gene panel including 450 genes related to monogenic forms of ID and autistic spectrum disorders (gene list is available upon request). After sonication (Covaris, Woburn, MA,

USA), library preparation was performed with Agilent SureSelectXT following manufacturer’s recommendations (Agilent Technologies, Santa Clara, CA, USA). 2x75bp paired-end sequencing was

performed on a NextSeq500 (Illumina, San Diego, CA, USA). Genomic alignment against the hg19/GRCh37 assembly and variant calling were, respectively, done with BWA-MEM v.0.7.12 and GATK

HaplotypeCaller v.3.4 (Broad Institute, Boston, MA, USA). Mean depth of coverage was 320 × with 98.5% of the target bases above 40 ×. Only highly confident variants were kept for analysis

(total depth >9; alternative allele depth >4; no strand bias; mosaicism >10%). Rare variants were considered as having a frequency <1% in public databases (ExAC, EVS, and 1000G).

Sanger sequencing was used to confirm variations that would possibly affect protein function. CLINICAL DESCRIPTION The first pregnancy of the couple ended in a miscarriage. PATIENT 1 This

girl was the first sib born at term after an uneventful pregnancy; delivery was normal. At birth, weight was 3470 g (50th centile), length was 50.5 cm (50th centile), and occipital frontal

circumference (OFC) was 33.5 cm (10–25th centile). Hypotonia, developmental delay, and strabismus were noted since the first months of life. At the age of 23 months, she started sitting

unsupported. Clinical examination showed frontal bossing, mild midface hypoplasia (Figures 1b and c), global hypotonia with normal reflexes, and poor eye contact. Standing with support was

achieved at the age of 3.5 years. EEG was normal at 16 months. At the age of 5.5 years, OFC was 50 cm (25–50th centile), height was 104.5 cm (25th centile), and weight was 17 kg (25th

centile). Global hypotonia, involuntary stereotypic movements, poor eye contact and social interactions, normal reflexes, and loose joints were noted. Speech was absent. Brain magnetic

resonance imaging (MRI), performed at 18 months of age, showed mild cerebral atrophy and thin corpus callosum (Figures 1d and e). Brain magnetic resonance spectroscopy (MRS) was normal.

Auditory evoked potential, fundus oculi examination, electroretinogram, and electromyography were normal. Heart and abdominal ultrasounds scans (USS) were normal. Genetic and metabolic

assessment was normal, including standard karyotype, array-CGH; _MECP2_ screening by sequencing and MLPA (multiple ligation probe amplification); methylation analysis for Prader–Willi

syndrome, search of homozygous _SMN1_ deletion by QMPSF (quantitative multiplex PCR of short fluorescent fragments) for spinal muscular atrophy, Southern blot for Steinert’s disease and

Fragile X syndrome; liquid chromatography-tandem mass spectrometry (LC-MS/MS) for plasma and urine amino acids, gas chromatography-mass spectrometry (GC-MS) for urine organic acids and

plasma very-long-chain fatty acids; screening for congenital disorders of glycosylation (Western blot). Blood levels of lactate, pyruvate (enzymatic spectrophotometry), and creatine kinase

(CK; measured according to the International Federation of Clinical Chemistry) were also normal. PATIENT 2 This boy was the second sib, born at 36 weeks of gestation after uneventful

pregnancy and delivery. At birth, weight was 2550 g (25th centile), length was 47 cm (25–50th centile), and OFC was 32 cm (10–25th centile). Developmental delay and hypotonia were noted. At

the age of 18 months, clinical examination showed frontal bossing (Figures 1f and g) and left transverse palmar crease. He could not sit unsupported and had hypotonia and stereotypic

movements (he used to look frequently at his hands and feet). At the age of 3 years, OFC was 50 cm (50th centile), height was 95.5 cm (50th centile), and weight was 14.8 kg (75th centile).

He was able to sit with support, but not to stand. Hypotonia, normal reflexes, and hand stereotypic movements were noted. Eye contact was present. No seizures were reported. Speech was

absent. Brain MRI and MRS, heart and abdominal USS were normal. CK plasma level was normal as were ammonia, LC-MS/MS for plasma amino acids, GC-MS for plasma very-long-chain fatty acids and

urine organic acids. Urine mucopolysaccharides (electrophoresis), oligosaccharides (thin-layer chromatography), sialic acid (MS/MS), urinary screening for adenylosuccinate lyase deficiency

(analysis of succinyl-AICA riboside by thin-layer chromatography) and plasma and urinary screening for creatine metabolism deficiencies (MS/MS) were normal. RESULTS Fourteen rare variants

were identified including only one homozygous missense transversion of _GRIN1_ in both siblings (hg19 chr9:g.140051128G>C). According to the longest isoform (NM_001185090.1) the

nomenclature of this variant is c.742G>C and the expected protein change is (NP_001172019.1) p.(Asp248His). In this paper, however, we will rather use the NM_007327.3 isoform to allow

comparison with previous reports (NM_007327.3 c.679G>C and NP_015566 p.Asp227His). It was not previously reported (PubMed, HGMD) and was absent from public databases of control

individuals (Exome Variant Server, 1000 genomes and ExAC) in patients. _In silico_ prediction tools for pathogenicity were in favor of a deleterious effect (Grantham=81, SIFT=1, and

Polyphen-2=1). Sanger sequencing confirmed the homozygous variant in both affected siblings and showed that both unaffected parents were heterozygous carriers (Figure 1a). The variant was

submitted to the specific _GRIN1_ LOVD database (variant ID 0000129232). DISCUSSION The c.679G>C _GRIN1_ transversion was the only homozygous rare variant found in the two affected

children and at the heterozygous state in the parents, who were healthy. The variant affects a rather conserved asparagine residue in the Zn2+ binding site, located within the aminoterminal

domain of the gene and is predicted to be deleterious by SIFT and Polyphen-2 (Figure 2a). Our patients have phenotypic similarities with two other sibs recently reported, who had a different

homozygous missense variant c.649C>T (p.(Arg217Trp)), located nearby within the aminoterminal domain of _GRIN1_ (Figure 2b).5 Patients from the two families share psychomotor delay,

hypotonia, and profound ID, as well as autistic features such as hand stereotypies (Table 1). Interestingly, they did not experience seizures, which is in strong contrast with patients

carrying _de novo_ heterozygous missense variants, who frequently had early onset drug-resistant epilepsy (70%, _n_=16/23). In contrast to the two homozygous missense variants, those

heterozygous variants cluster in and around the transmembrane domains, which is highly conserved among different species. _In vitro_ functional experiments showed that most _de novo_

heterozygous variants lead to a dominant negative effect, whereas the c.649C>T variant showed impaired activation due to tonic inhibition of _GRIN1_Arg217Trp-_GRIN2A_ receptors.5 In

addition, another family was reported, including three sibs affected with neonatal epileptic encephalopathy with intractable seizures leading to death between 5 days and 5 months. The

affected sibs had a homozygous truncating variant of _GRIN1_ c.1666C>T (p.(Gln556*)), which was found at the heterozygous state in the unaffected parents. This suggests that heterozygous

truncating variants of _GRIN1_ apparently do not result in a neurological phenotype. Variants in the Zn2+ binding domain appear to be rare in genes encoding NMDAR subunits. The heterozygous

missense variant p.(Ala243Val) in _GRIN2A_ has been shown to significantly reduce high-affinity Zn2+ inhibition, mediating a gain-of-function.6 For the c.679G>C variant, which is located

in GRIN1 in a position similar to p.(Ala243Val) in GRIN2A, we could rather expect a loss-of-function mechanism. Functional studies, however, would be useful to answer this question. The fact

that the phenotypic expression of deleterious variants in a given gene may be related to different modes of transmission has been reported for several genes. _LMNA_ is probably the most

famous example. This gene is associated with about 15 different diseases including Emery–Dreifuss muscular dystrophy, limb-girdle muscular dystrophy type 1B and Huntchinson–Gilford progeria

that mostly follow an autosomal-dominant mode of inheritance, and Charcot–Marie–Tooth disease type 2B1 that is an autosomal recessive disorder.7 In the field of epilepsy, such a situation

has also been observed for several genes. Some variants in _POLG_ are found in families with autosomal-dominant progressive external ophtalmoplegia, whereas other variants cause a range of

autosomal-recessive conditions including Alpers syndrome, mitochondrial neurogastrointestinal encephalopathy, or sensory ataxic neuropathy, dysarthria, and ophtalmoparesis syndrome.8

Recently, some missense variants of _RELN_ have been shown to cause autosomal-dominant lateral temporal epilepsy, whereas other variants were previously found in patients affected with

autosomal-recessive lissencephaly with cerebellar hypoplasia.9, 10 Homozygous or biallelic variants have not been reported so far for genes encoding other subunits of NMDA receptors, such as

_GRIN2A_ and _GRIN2B_, whose heterozygous variants cause various ranges of neurodevelopmental disorders with epilepsy.11, 12, 13, 14 The present family together with previous reports

suggests that heterozygous variants located in the aminoterminal domain may have a hypomorphic effect, whereas heterozygous variants located in the transmembrane region may rather lead to a

gain-of-function. In conclusion, the c.679G>C (p.Asp227His) transversion is likely to be a hypomorphic variant, which is pathogenic at the homozygous state, whereas the functionality of

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Neurol_ 2014; 75: 147–154. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the NGS platform of Lyon University Hospital and the bioinformaticians (Claire Bardel

and Pierre Antoine Rollat-Farnier), as well as Raphaelle Lamy for technical assistance. AUTHOR INFORMATION Author notes * Massimiliano Rossi and Nicolas Chatron: These authors contributed

equally to this work. AUTHORS AND AFFILIATIONS * Department of Genetics, Lyon University Hospitals, Lyon, France Massimiliano Rossi, Nicolas Chatron, Audrey Labalme, Patrick Edery, Damien

Sanlaville & Gaetan Lesca * Lyon Neuroscience Research Centre, CNRS UMR5292, INSERM U1028, Lyon, France Massimiliano Rossi, Nicolas Chatron, Patrick Edery, Damien Sanlaville & Gaetan

Lesca * Department of Pediatric Neurology, Lyon University Hospitals, Lyon, France Dorothée Ville, Maryline Carneiro & Vincent des Portes * Claude Bernard Lyon I University, Lyon,

France Patrick Edery, Vincent des Portes, Damien Sanlaville & Gaetan Lesca * Institute of Human Genetics, University of Leipzig Hospitals and Clinics, Leipzig, Germany Johannes R Lemke

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INTERESTS The authors declare no conflict of interest. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Rossi, M., Chatron, N., Labalme, A. _et al._

Novel homozygous missense variant of _GRIN1_ in two sibs with intellectual disability and autistic features without epilepsy. _Eur J Hum Genet_ 25, 376–380 (2017).

https://doi.org/10.1038/ejhg.2016.163 Download citation * Received: 09 May 2016 * Revised: 18 October 2016 * Accepted: 25 October 2016 * Published: 04 January 2017 * Issue Date: March 2017 *

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