MD Stefanie Brock

Annie Laquerriere

MD Florent Marguet

PhD Scott J Myers

Yuan Hongjie

MD Diana Baralle

MD Tim Vanderhasselt

MD Katrien Stouffs

MD Kathelijn Keymolen

Sukhan Kim

MD Annie Laquerrière

MD, PhD Hongjie Yuan

James P Allen

Gil Shaulsky

MD Jamel Chelly

MD Pascale Marcorelles

MD Jacqueline Aziza

PhD Laurent Villard

MSc Elise Sacaze

MD Marie Claire De Wit

MD Martina Wilke

MD Grazia Mancini

MD Ute Hehr

MBChB Derek Lim

BMBS, FRCP Sahar Mansour

PhD Stephen F Traynelis

MD Claire Beneteau

MD Marie Denis-Musquer

MD Anna C Jansen

MBChB, DPhil Andrew E Fry

MD Nadia Bahi-Buisson

Overlapping cortical malformations in patients with pathogenic variants in GRIN1 and GRIN2B

Keywords: /jmedgenet GRIN1, GRIN2B, NMDAR, malformations of cortical development, polymicrogyria

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INTRODUCTION

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated cation channels mediating excitatory synaptic transmission. The NMDAR is a heteromeric complex typically composed of two GluN1 subunits (encoded by GRIN1) and two GluN2 subunits (encoded by GRIN2A-D). The combination of different subunits influences receptor function. [START_REF] Cull-Candy | NMDA receptor subunits: diversity, development and disease[END_REF][START_REF] Traynelis | Glutamate Receptor Ion Channels: Structure, Regulation, and Function[END_REF] NMDAR subunits contain a discontinuous ligand binding domain (S1 and S2), an intracellular re-entrant pore loop (M2) and three transmembrane domains (M1, M3 and M4). [START_REF] Sobolevsky | Molecular rearrangements of the extracellular vestibule in NMDAR channels during gating[END_REF] NMDAR subunits exhibit different spatiotemporal expression patterns. The essential glycine-binding GluN1 subunit is highly and ubiquitously expressed in the fetal and adult brain. [START_REF] Traynelis | Glutamate Receptor Ion Channels: Structure, Regulation, and Function[END_REF] In contrast, the glutamate-binding subunit GluN2B (encoded by GRIN2B) is highly expressed prenatally with progressive limitation to the forebrain, before being replaced postnatally by GluN2A (encoded by GRIN2A). [START_REF] Mckay | The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences[END_REF] Pathogenic variants in GRIN1 and GRIN2B have been described in individuals with a spectrum of neurological phenotypes. In individuals with heterozygous pathogenic variants, common features include developmental delay, intellectual disability, epilepsy, cortical visual impairment, neurobehavioral problems and movement disorders. [START_REF] Endele | Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes[END_REF][START_REF] Hamdan | Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability[END_REF][START_REF] Lemke | GRIN2B mutations in West syndrome and intellectual disability with focal epilepsy[END_REF][START_REF] Ohba | GRIN1 mutations cause encephalopathy with infantile-onset epilepsy, and hyperkinetic and stereotyped movement disorders[END_REF][START_REF] Lemke | Delineating the GRIN1 phenotypic spectrum: A distinct genetic NMDA receptor encephalopathy[END_REF][START_REF] Xiangwei | De Novo Mutations and Rare Variants Occurring in NMDA Receptors[END_REF] Homozygous variants have only rarely been reported in GRIN1. These include four individuals from two different families with homozygous missense variants, developmental delay, movement disorders but without seizures [START_REF] Lemke | Delineating the GRIN1 phenotypic spectrum: A distinct genetic NMDA receptor encephalopathy[END_REF][START_REF] Rossi | Novel homozygous missense variant of GRIN1 in two sibs with intellectual disability and autistic features without epilepsy[END_REF]; and four individuals from two families with homozygous truncating variants with severe neonatal epileptic encephalopathy. [START_REF] Lemke | Delineating the GRIN1 phenotypic spectrum: A distinct genetic NMDA receptor encephalopathy[END_REF][START_REF] Blakes | A homozygous GRIN1 null variant causes a more severe phenotype of early infantile epileptic encephalopathy[END_REF] Homozygous variants in GRIN2B have not been reported yet. To date, 12 patients harbouring de novo heterozygous pathogenic variants in GRIN1 and six patients with pathogenic variants in GRIN2B have been reported to have MCDs. [START_REF] Fry | De novo mutations in GRIN1 cause extensive bilateral polymicrogyria[END_REF][START_REF] Nishimura | Arthrogryposis multiplex congenita with polymicrogyria and infantile encephalopathy caused by a novel GRIN1 variant[END_REF][START_REF] Platzer | GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects[END_REF] This subset of patients accounts for approximately 10-15% of all reported individuals with pathogenic variants in either GRIN1 or GRIN2B. [START_REF] Platzer | GRIN2B-Related Neurodevelopmental Disorder[END_REF][START_REF] Platzer | GRIN1-Related Neurodevelopmental Disorder[END_REF] GRIN1 or GRIN2B are characterized by variable sulcal depth and/or orientation with normal cortical thickness. This was described as 'dysgyria', the generic term for abnormal cerebral cortex, although the malformation seems to closely resemble polymicrogyria. [START_REF] Fry | De novo mutations in GRIN1 cause extensive bilateral polymicrogyria[END_REF][START_REF] Severino | 19 Deciphering Developmental Disorders Study. Large-scale discovery of novel genetic causes of developmental disorders[END_REF] However, histopathological evidence has not been available from previous patients to confirm this. Associated brain malformations such as dysmorphic basal ganglia, hypoplastic corpus callosum and hippocampal dysplasia have been reported. [START_REF] Fry | De novo mutations in GRIN1 cause extensive bilateral polymicrogyria[END_REF][START_REF] Platzer | GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects[END_REF] The pattern of brain malformations associated with NMDAR-associated MCDs has been noted to resemble that of tubulinopathies. [START_REF] Fry | De novo mutations in GRIN1 cause extensive bilateral polymicrogyria[END_REF][START_REF] Platzer | GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects[END_REF] To provide further insights into the clinical and radiological features of NMDARassociated MCDs, we report four patients with pathogenic heterozygous variants in GRIN1 and three patients with heterozygous variants in GRIN2B who all presented with complex brain malformations, including dysgyria, and malformations of the hippocampus and basal ganglia. In addition, we present a single patient with a homozygous variant in GRIN1 associated with a simplified gyral pattern (SGP). The neuropathological data, which were available for two foetuses with de novo heterozygous GRIN1 variants and the neonate with the homozygous GRIN1 variant, contribute to our understanding of the underlying pathophysiological mechanisms causing the phenotypic features of NMDAR-associated

MCD. C o n f i d e n t i a l : F o r R e v i e w O n l y

MATERIALS AND METHODS

Patient selection

The patients were identified through the Neuro-MIG Network (COST Action CA16118). The study was approved by the ethical committee of the UZ Brussel A detailed description of these methods is provided in the Supplementary Material. In summary, the p.Cys744Tyr and the p.Ala653Thr variants were introduced into a plasmid expression construct containing the human GluN1-1a cDNA (hereafter GluN1, based on NM_007327.3) and the p.Met818Thr introduced into human GluN2B cDNA (NM_000834.3) via a QuikChange mutagenesis protocol. The cRNAs for wild type and mutant forms of GluN1, were co-injected with wild-type human GluN2A cRNA and wild-type and mutant forms of GluN2B with wild-type human GluN1 cRNA into Xenopus laevis oocytes. The expressed receptors were functionally evaluated by two-electrode voltage clamp (TEVC) recordings for dose response to L-glutamate, glycine, Mg 2+ and Zn 2+ ions, and for proton sensitivity.

Electrophysiological analysis

Neuropathological studies

After complete autopsy, brains were fixed in a 10% formaldehyde-zinc buffer solution.

Macroscopic and microscopic examination was performed according to standardized procedures. Immunohistochemical studies were performed using markers for neuronal progenitors, migrating neurons and interneurons as well as for cortical layering (Supplementary material). Results were compared with age-matched controls aged 25, 30 and 39 gestational weeks, respectively.

RESULTS

Clinical features in MCD patients with pathogenic heterozygous variants in GRIN1 and

GRIN2B

We identified seven unrelated MCD patients (individuals 1-7) with de novo heterozygous pathogenic variants in either GRIN1 or GRIN2B. Clinical findings for individuals 1 to 7 are summarized in Table 1. Two individuals with GRIN1 variants (individuals 1 and 2) and the three individuals with GRIN2B variants presented with microcephaly, severe developmental delay and epilepsy. The age of seizure onset ranged from the neonatal period to 2 years of age. Seizure types were variable, and refractory to treatment in two individuals. Individuals 3 and 4 were foetuses with variants in GRIN1 that underwent termination of pregnancy. Autopsy and neuropathological findings from individuals 3 and 4 are described in the section below.

In total, 25 MCD patients with heterozygous variants in either GRIN1 or GRIN2B have now been reported in this series and in the wider literature. We reviewed the available clinical information for these 25 patients (Supplementary material, Tables S1 andS2). GRIN1 or GRIN2B MCD patients had overlapping severe clinical phenotypes. Common features in this combined cohort included severe or profound developmental delay or intellectual disability (13/13 in GRIN1, 9/9 in GRIN2B), epilepsy (13/13 in GRIN1, 9/9 in GRIN2B), microcephaly (12/14 in GRIN1, 5/6 in GRIN2B), spastic quadriparesis (9/12 in GRIN1, 2/3 in GRIN2B) and cortical visual impairment (10/11 in GRIN1, 4/5 in GRIN2B). Movement disorders were present in 3/5 individuals with variants in GRIN1 but have not been reported in individuals with variants in GRIN2B. The age of onset of epilepsy was variable across this combined cohort ranging from the neonatal period to 1 year for GRIN1, and from 10 weeks to 2 years 3 months for GRIN2B. Epilepsy phenotypes were also variable in the combined cohort,

Brain imaging in MCD patients with pathogenic variants in GRIN1 and GRIN2B

Brain magnetic resonance imaging (MRI) was performed in all seven individuals with heterozygous variants in GRIN1 or GRIN2B in our series between 29 weeks gestation and three years of age (Table 1, Figure 1), except for individual 3 (in which only prenatal ultrasound was performed). All individuals with variants in GRIN1 presented with abnormal gyration. The two living GRIN1 patients (individuals 1 and 2) exhibited bilateral dysgyria, predominantly affecting the frontal regions. Among the GRIN2B patients, individuals 5 and 6 demonstrated diffuse bilateral dysgyria while individual 7 had bilateral but asymmetric dysgyria predominantly affecting the left hemisphere.

We reviewed the available neuroradiology for all 25 heterozygous NMDAR MCD patients (Supplementary material, Tables S1 andS2). We found that the MRI phenotypes of GRIN1-and GRIN2B patients were very similar (Supplementary material, Table S3).

Common MRI features in the combined cohort included diffuse bilateral dysgyria often with some posterior sparing, reduced white matter, enlarged lateral ventricles, hippocampal dysplasia, hypoplastic corpus callosum, and enlarged or dysmorphic basal ganglia. The brainstem was normal on imaging in all GRIN1 and GRIN2B patients, except for mildly reduced myelination of the brainstem in individual 1 in this study. The cerebellum was abnormal in 3 individuals with variants in GRIN1, but normal in all individuals with variants in GRIN2B. It was not possible to distinguish GRIN1-and GRIN2B-related MCDs based on the radiological findings alone.

Molecular findings in MCD patients with pathogenic variants in GRIN1 and GRIN2B

We identified three GRIN1 and two GRIN2B variants that have not previously been reported.

The variants were classified as pathogenic or likely pathogenic (Supplementary material, Tables S4 andS5). The p.(Ser553Leu) GRIN1 variant in individual 1 is a pathogenic variant previously reported in an unrelated MCD patient. [START_REF] Fry | De novo mutations in GRIN1 cause extensive bilateral polymicrogyria[END_REF] The p.(Met818Thr) GRIN2B variant in individual 7 has previously been reported in a non-MCD patient. [START_REF] Platzer | GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects[END_REF] The seven variants were located in the same regions where previous MCD-associated NMDAR variants have clustered (Figure 2).

To investigate the impact of two GRIN1 variants and one GRIN2B variant, functional studies were performed. The full results are given in the supplementary data section 3.

Compared to wild type GluN1 the concentrations that elicit a half-maximal current (EC 50 ) for GluN1-C744Y, GluN1-A653T and GluN2B-M818T were reduced for L-glutamate and for glycine, indicating these agonists have significantly greater potency (i.e. produce responses at lower agonist concentrations). Mutant receptors were also less sensitive to inhibition by protons. Expression of the variants GluN1-C744Y and GluN1-A653T subunit protein does not appear to be altered substantially as the expressed levels of both surface and total receptor protein were modest. Overall, these in vitro electrophysiological and biochemical analyses suggest that the three variants cause a gain of function. Macroscopic and histological examination of all viscera was normal in both cases. Both patients had reduced brain weight for gestational age (<<3 rd percentile). [22] On external macroscopic brain examination of individual 3, the brain surface appeared smooth with a dimple-shaped Sylvian fissure (Figure 3A and3B). On sagittal and coronal sections, the ventricles were enlarged and the corpus callosum was absent. Histologically, the thin cortical ribbon had a diffuse festooned-like appearance suggestive of polymicrogyria under a thick and irregular molecular layer containing some immature neuronal heterotopias, and covered by a persistent transient external granular cell layer (Figure 3C). Nodular heterotopias were also noted in the periventricular regions close to the germinative zone of the dorsal telencephalon, to the ganglionic eminences and within the basal ganglia (Figure 3D). The

C o n f i d e n t i a l : F o r R e v i e w O n l y
infratentorial structures were free of lesions. External macroscopic brain examination in individual 4 revealed diffuse polymicrogyria (Figure 3E). The olfactory tracts were absent.

The Sylvian fissure was short, vertically oriented and prematurely closed (closure normally occurs between 36 and 38 weeks gestation). The corpus callosum was hypoplastic in its posterior part (Figure 3E). The lateral ventricles were moderately dilated. The hippocampi appeared to be malrotated (Figure 3F). Histologically, the cortical ribbon was replaced by diffuse and poorly laminated polymicrogyria (Figure 3G) covered by a glia limitans which was focally discontinuous with small foci of overmigration. The internal capsule was hypoplastic, the ganglionic eminences had already regressed and the basal ganglia were dysplastic, as were the thalami which were formed by the juxtaposition of nodules. Small confetti-like heterotopic nodules were observed throughout the intermediate zone (Figure 3H).

The cerebellar vermis was hypoplastic but the brainstem and the spinal cord were normal.

We compared the clinical and neuropathological findings of individuals 3 and 4 to individual 8 with novel homozygous truncating variants (p.Tyr474*) in GRIN1 inherited from asymptomatic heterozygous carrier parents. Shortly after birth, individual 8 developed seizures evolving to status epilepticus. The patient died at 6 days of age. Brain imaging was not performed. Individual 8 had microcephaly with a SGP without tertiary sulci on macroscopic examination (Supplementary material, Figure S1). The corpus callosum was hypoplastic. Histologically, multiple foci of neuronal depletion with loss of lamination and abnormal persistence of the external transient granular cell layer in some areas were observed.

In the hippocampus, the dentate gyrus was dysmorphic and discontinuous. Small germinolysis pseudo-cysts were observed along the ventricular walls (Supplementary material, Figure S1).

Measurement of the cortical thickness was 1.97 mm, which does not significantly differ from cortical thickness measured on several control cortical plates between 30 and 38 WG (1.70 to 1.90 mm). These data are not in favour of lissencephaly with thin or thick cortex.

C o n f i d e n t i a l : F o r R e v i e w O n l y
Immunohistochemistry revealed less numerous SOX2 and PAX6-positive proliferating radial glial cells in the ventricular and outer subventricular zones of individual 3 than observed in the control brain aged 24-25 WG (Supplementary material, Table S7). MAP2 antibody which immunolabels radially migrating neurons and pyramidal neurons of layers III and V did not allow for identifying any immunoreactive neuron in the micropolygyric cortex of individual 3, these neurons being observed within the periventricular heterotopic nodules (Figure 4A). In individuals 4 and 8, very few MAP2-positive neurons were identified in the cortical plate.

These neurons were misoriented, dysmorphic and/or immature (Figure 4B, C andD). CTIP2 and SATB2-positive neurons were either absent or mislocalized (Figure 4E-H). GABAergic interneurons were aberrantly located or almost absent, as were calretininergic interneurons NMDAR variants with GOF effects have also been observed in patients in which no MCDs were reported. [START_REF] Lemke | GRIN2B mutations in West syndrome and intellectual disability with focal epilepsy[END_REF][START_REF] Platzer | GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects[END_REF][START_REF] Swanger | Mechanistic Insight into NMDA Receptor Dysregulation by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains[END_REF]29] These observations raise the possibility that a patient's genetic background or non-genetic events (e.g. viral infection or hypoxic-ischemic injury) may be contributing to the pathogenesis of NMDAR-MCDs. Another explanation can be subtle changes of cortical architecture that might be missed on imaging studies, as has been shown

in individual 8 with SGP and homozygous LOF variants. Of note, the homozygous variants in individual 8 were truncating variants. These are likely to cause complete LOF of GRIN1, as has been reported in three siblings and one additional individual with a similar clinical course of neonatal epileptic encephalopathy with homozygous truncating variants in GRIN1. [START_REF] Lemke | Delineating the GRIN1 phenotypic spectrum: A distinct genetic NMDA receptor encephalopathy[END_REF][START_REF] Blakes | A homozygous GRIN1 null variant causes a more severe phenotype of early infantile epileptic encephalopathy[END_REF] Homozygous truncating variants causing complete absence of the protein might therefore be at the most severe end of clinical and neuropathological features of LOF variants in in GRIN1.

Thus, the growing evidence for a genotype-phenotype correlation suggests that intrinsic properties of specific NMDAR variants need to be considered as well. [START_REF] Xiangwei | De Novo Mutations and Rare Variants Occurring in NMDA Receptors[END_REF] is not supported. Finally, the lack of polymicrogyria in the individual with biallelic LOF variants supports the hypothesis that clinical, histological and pathophysiological differences between gain-and loss of function GRIN-variants exist.

The seven heterozygous NMDAR variants reported in our series were located in the same regions as previously reported MCD-associated variants. MCD-associated GRIN1 variants (Figure 1) have clustered in the S1-M1 linker (pre-M1) region, the Lurcher motif in the M3 region, and the S2 domain of GluN1. [START_REF] Fry | De novo mutations in GRIN1 cause extensive bilateral polymicrogyria[END_REF] MCD-associated GRIN2B variants have clustered in the M3, S2 and M4 domains of GluN2B. [START_REF] Platzer | GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects[END_REF] Interestingly, while non-MCD variants in GRIN1 and GRIN2B have been described, no MCD-associated GRIN1 variants have yet been reported in the GluN1 M4 helix (a region where around half of non-MCD GRIN1 variants have been located [START_REF] Lemke | GRIN2B mutations in West syndrome and intellectual disability with focal epilepsy[END_REF][START_REF] Lemke | Delineating the GRIN1 phenotypic spectrum: A distinct genetic NMDA receptor encephalopathy[END_REF]) or the pre-M1 region of GluN2B. This may reflect functional differences between these two domains between GluN1 and GluN2 subunits.

In the patients reported to date, the spectrum of NMDAR variants, including recurrent variants, such as the p.(Ser553Leu) GRIN1 variant in individual 1, is different from the variants found in non-MCD patients. An exception to this observation has been the recurrent p.(Met818Thr) in GRIN2B, located in the GluN2B M4 helix, which has been reported in both an MCD patient (individual 7 in this paper) and a previously reported non-MCD patient. [START_REF] Platzer | GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects[END_REF] Neuroimaging data from the non-MCD patient has not been available for review. shown to increase the potency of glutamate and reduce proton inhibition. [START_REF] Sullivan | Identification of two cysteine residues that are required for redox modulation of the NMDA subtype of glutamate receptor[END_REF] The Cys744-Cys798 disulphide bond has been shown to modulate behavior of NMDAR in response to redox environment. [START_REF] Takahashi | Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif[END_REF][START_REF] Brimecombe | Functional consequences of NR2 subunit composition in single recombinant N-methyl-D-aspartate receptors[END_REF] In pathological situations (e.g. following stroke or hypoxia) the brain environment becomes more reductive which favors free thiol over disulphide formation. [START_REF] Takahashi | Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif[END_REF] It has been proposed that the Cys744-Cys798 bond is a 'molecular oxygen sensor' in the brain, responding to low pO2 levels by controlling how much NO can inhibit NMDA function. [START_REF] Takahashi | Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif[END_REF] There is evidence that disruption of NMDA signaling can influence the proliferation, migration and maturation of neuronal progenitor cells and post-mitotic neurons. [START_REF] Jiang | The GluN2B subunit of N-methy-D-asparate receptor regulates the radial migration of cortical neurons in vivo[END_REF][START_REF] Bell | Disruption of GRIN2B Impairs Differentiation in Human Neurons[END_REF] A higher prevalence of microcephaly in MCD patients together with increased potency of the NMDAR during electrophysiological studies and reduced neuronal density on histology suggest increased NMDAR signaling leading to excitotoxic cell death may contribute to the increased frequency of microcephaly. This also suggests that the impact on proliferation or survival of neurons is more common or more severe in MCD-associated NMDAR variants.

Alternatively, it has been noted that MCD-associated NMDAR variants tend to have GOF effects. [START_REF] Fry | De novo mutations in GRIN1 cause extensive bilateral polymicrogyria[END_REF] This is consistent with animal models of NMDAR-agonist induced polymicrogyria. [START_REF] Marret | Effect of ibotenate on brain development: an excitotoxic mouse model of microgyria and posthypoxic-like lesions[END_REF][START_REF] Takano | Radial and tangential neuronal migration disorder in ibotenate-induced cortical lesions in hamsters: immunohistochemical study of reelin, vimentin, and calretinin[END_REF][START_REF] Takano | Increased expression of GAP43 in interneurons in a rat model of experimental polymicrogyria[END_REF] In conclusion, the MCD patients with GRIN1 and GRIN2B variants reported to date have overlapping phenotypic features, consisting of early presentation of global developmental delay, epilepsy and microcephaly. Brain imaging mostly shows severe dysgyria, histologically in line with polymicrogyria in two cases, and often with associated brain malformations including dysmorphic basal ganglia and hippocampi. These findings extend our understanding of the "NMDARopathy" spectrum. Our neuropathological findings In Glycine dose response studies: The recording solution for these studies was similar as for the Lglutamate studies. The oocyte membrane potential was clamped at -40 mV. After a steady baseline was obtained, oocytes were perfused with increasing concentrations of glycine (seven concentrations selected from 0.003, 0.01, 0.03, 0. -Lactamase Assay: HEK cells were plated in 96-well plates and transiently transfected with cDNA encoding GluN2A-WT and -lac-GluN1-WT or -lac-GluN1-C744Y or -lac-GluN1-A653T. Eight wells were transfected with surface and total expression activities measured in 4 wells each 24 hr post transfection. The ratio of surface (unlysed cells) to total (lysed cells) -lactamase expression was measured for the -lac-GluN1-C744Y and -lac-GluN1-A653T compared to -lac-GluN1-WT. See PMID 27839871 for further information.

Equation 1: Response = 100 / ( ( 1 + EC50 / [agonist] ) nH ) where EC50 is the agonist concentration that elicited the half maximal response, and nH is the Hill slope.

Equation 2: Response = (100 -minimum) / (1 + ([concentration] / IC50) nH ) + minimum where minimum is the residual percent response in saturating concentration (constrained to > 0) of the experimental compounds, IC50 is the concentration of inhibitor that causes half maximal inhibition, and nH is the Hill slope.

Statistical comparisons: For analysis of the IC50 or EC50 results, the log values of the IC50's or EC50's were used for comparison. If the 95% confidence intervals of WT and variant results for each assay do not overlap, a fold effect was then calculated with positive and negative fold values indicating increased or decreased effect on receptor activity, respectively, for that assay measurement.

b. RESULTS

https formalin-zinc buffer solution for at least one month. Brain growth and macroscopic assessment of brain maturation including gyration were evaluated according to the criteria of Guihard-Costa and Larroche and the atlas of Feess-Higgins and Larroche (1990,1987).

Eight-micrometer sections obtained from paraffin-embedded tissues were stained using Haematoxylin-Eosin. Immunohistochemical procedures included a microwave pretreatment protocol to aid antigen retrieval (pre-treatment CC1 kit, Ventana Medical Systems Inc, Tucson AZ). Incubations with the primary antibodies listed in supplementary Table 1 were performed for 32 or 44 minutes at room temperature using the Ventana Benchmark

(

  B.U.N.143201214360). Three of the patients (individuals 1, 2 and 7) were part of the Deciphering Developmental Disorders (DDD) study.[19] The DDD study was approved by the Cambridge South Research Ethics Committee (10/H0305/83). Clinical data was collected through clinical examination and review of medical records. Imaging data was reviewed by the authors (S.B., A.E.F., N.B-B.).Genetic analysisDNA preparation and genetic investigation was undertaken with informed consent from the parents or guardians of the patients. DNA was derived from saliva (individuals 1, 2 and 7), blood (individuals 5, 6 and 8) and by amniocentesis (individuals 3 and 4). DNA was extracted using standard protocols. Whole-exome sequencing (WES) was performed in individuals 1-4, and 6 and 7. Individuals 5 and 8 were tested by next-generation sequencing (NGS) multi-gene panels. Sanger sequencing was used in all individuals except for individual 6 to validate the variants and to test parents. The positions of variants are based on transcripts NM_007327.3 for GRIN1 and NM_000834.3 for GRIN2B. Variants were classified using American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG-AMP) criteria.[START_REF] Ellard | ACGS 2019 Best Practice Guidelines for Variant Classification[END_REF] 21]

C

  o n f i d e n t i a l : F o r R e v i e w O n l y 13

Individuals 3 and 4 ,

 4 harboring de novo heterozygous variants in GRIN1, underwent termination of pregnancy at 25 and 30 weeks gestation, respectively, according to the French law, due to severe brain malformations identified on ultrasound imaging. In individual 3, ultrasound (US) examination performed at 23 WG revealed agenesis of the corpus callosum and of the septum pellucidum. Severe deformations of the extremities consisting in bilateral camptodactyly and equinovarus talipes were noted. Based on these findings a medical termination of the pregnancy was proposed and accepted by both parents. In individual 4, US performed at 24 WG identified mild ventricular dilatation. At 26 WG, control US revealed diffuse dysgyria and MRI performed at 29 WG allowed the detection of absent primary fissures and secondary sulci suggestive of lissencephaly. Neither deformities of the limbs nor polyhydramnios were identified. Both individuals showed mild facial particularities consisting of prominent philtrum, broad nasal bridge with anteverted nostrils and flat face.

Figure 1 :

 1 Figure 1: Brain imaging findings of individuals with heterozygous pathogenic variants

Figure 2 :

 2 Figure 2: Distribution of variants in linear model and 3D protein structure for GRIN1

Figure 3 :brain morphological characteristics from individuals 3 and 4 Individual 3 :

 343 Figure 3: Distinctive brain morphological characteristics from individuals 3 and 4

Figure 4 :findings in individuals 3 , 4 and 8 A

 438 Figure 4: Immunohistochemical findings in individuals 3, 4 and 8

  Abbreviations: BG -basal ganglia, CC -corpus callosum, H&E -haematoxylin-eosin

Figure 1 :

 1 Figure 1: Brain imaging findings of individuals with heterozygous pathogenic variants in GRIN1 and GRIN2B. Individual 1 has diffuse dysgyria, hypoplastic corpus callosum (arrow) and dysmorphic hippocampi (arrowhead) (A-C). Individual 2 has bilateral dysgyria, hypoplastic corpus callosum (arrow) and dysmorphic basal ganglia (asterisk) (D-F). Prenatal ultrasound in individual 3 at 25 gestational weeks shows absent corpus callosum (arrow) and dilated third ventricle (arrowhead) (G-H). Individual 4 has diffuse dysgyria and enlarged lateral ventricles on prenatal MRI at 29 gestational weeks (I-J). Individual 5 presents with bilateral dysgyria, enlarged basal ganglia (asterisk) and hypoplastic corpus callosum (arrow) (K-M). Individual 6 has bilateral symmetric dysgyria and dysmorphic hippocampi (arrowhead) (N-P). Individual 7 has asymmetric dysgyria of the left hemisphere, dysmorphic basal ganglia (asterisk) and corpus callosum (arrow) (Q-S). 190x338mm (96 x 96 DPI)

Figure 2 :

 2 Figure 2: Distribution of variants in linear model and 3D protein structure for GRIN1 (A, C) and GRIN2B (B, D). In the linear model, clustering of MCD-associated variants (top) and non-MCD associated variants (bottom, blue triangles) is highlighted. Functional domains are highlighted in the 3D model: blue -helices, greensheets. Reported variants from this cohort are written in orange, variants reported in the literature are blue, the recurring variant in the literature and our cohort is green. The 3D structure is based on Swiss Models NMDZ1_HUMAN Glutamate receptor ionotropic, NMDA1 Q05586 and NMDE2_HUMAN Q13224 Glutamate receptor ionotropic, NMDA 2B.

Figure 3 :

 3 Figure 3: Distinctive brain morphological characteristics from individuals 3 and 4 Individual 3: A) External view of the left side of the brain, where primary fissures are lacking, with almost indiscernible Sylvian fissure (arrow) and marked temporal lobe hypoplasia (blue asterisk), B) compared to an age matched control brain, where the Sylvian fissure has a triangular shape (arrow). C) The brain surface is smooth with a relatively thick transient subpial granular cell layer which should have been disappeared from 22 WG (arrow) and diffuse micropolygyria forming multiple protrusions into the molecular layer [OM x 20].D) The cerebral mantle is particularly thin (half of the thickness of a normal cortical plate) and the deep intermediate zone contains numerous heterotopic nodules of various sizes (arrow) above the germinative zone of the dorsal telencephalon (asterisk) [OM x 20].Individual 4: E) Median supratentorial sagittal section revealing posterior hypoplasia of the corpus callosum (white arrow), F) and on coronal sections, supernumerary gyri (asterisks), areas of smoothened cortex covering polymicrogyria (black arrows) and malrotated hippocampus (white arrow). G) Characteristic histological features of unlayered polymicrogyria [H&E, OM x 50], H) associated with confetti-like nodular heterotopias, measuring about 1.50 µ, distributed throughout the intermediate zone (arrow) [H&E, OM x 50].

Figure 4 :

 4 Figure 4: Immunohistochemical findings in individuals 3, 4 and 8 A) Strong MAP2 immunoreactivity in the periventricular heterotopic nodules in individual 3. B) Scattered dysmorphic MAP2-positive pyramidal neurons in the cortical plate (individual 4). C) Decreased density in MAP2-positive haphazardly dispersed pyramidal neurons in the cortical plate (individual 8), D) compared to the control cortical plate aged 39 WG in which neurons of layers II, III and V with predominance in layer V (arrow). E) Almost no SATB2-positive neurons in the cortex of individual 4, F) contrary to the control brain aged 30 WG where SATB2 positive neurons are observed mainly in layer II (arrow) and to a lesser extent in Layers III and IV. G) No CTIP2 immunoreactive neurons (individual 8), H) whereas in the control cortical plate, immunoreactive neurons are observed mainly in layer V (arrow). I) Few GABAergic interneurons dispersed in the micropolygyric cortex (individual 4), J) by comparison with the control brain where GABA antibody immunolabels interneurons of layers I, II (thin arrow) and III (thick arrow), K) and with scarce calretininergic interneurons in layer III (individual 8), L) conversely to the control, where calretinin antibody immunolabels the tangential superficial network and Cajal Retzius cells (arrow) in layer I, interneurons in layers II and III. Dark brown staining: specific, light brown: background staining; 25 WG: individual 3; 30 WG: individual 4; 39 WG: individual 8. Abbreviations: BG -basal ganglia, CC -corpus callosum, H&E -haematoxylin-eosin staining, OM -original magnification.412x231mm (300 x 300 DPI)

O

  XT system. After incubation, slides were processed by means of the Ultraview Universal DAB detection kit (Ventana). A comparative semi-quantitative analysis of immunolabellings in the germinative and intermediate zones as well as in the different layers of the frontal cortex of affected and control patients was assessed and evaluated as follows: 0: no cell labelled; +: very few cells labelled; ++: moderate density of immunolabelled cells and +++: high density of immunolabelled cells. https://mc.manuscriptcentral.com/jmedgenet Journal of Medical Genetics C o n f i d e n t i a l : F o r R e v i e w

d. Supplementary figure 1 : 8 A 15 C

 1815 Main brain macroscopic and histological findings of individual ) Macroscopic view of right side of the neonate brain displaying delayed gyration with no tertiary gyri, broadened sulci and frontal lobe hypoplasia, B) compared to an age matched control brain, where the tertiary sulcation is in place. ) Histological view of the temporal cortex in which neuron depletion and dyslamination are observed underneath layer II [H&E, OM x 50], D) compared with the six-layered cortex of an age-matched control temporal cortex [same OM]. E) Histological view of the hippocampus showing an irregular and fragmented dentate nucleus (arrows), contrasting with a preserved pyramidal cell layer [H&E, OM x 50], F) compared with a normal hippocampus at the same age [H&E, OM x 50].

  and authorise the granting of this licence.

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  Basedon radiological appearance, the MCDs observed in patients with pathogenic variants in

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Table 1 .

 1 Clinical and imaging features of novel patients with variants in GRIN1 and GRIN2B

					Journal of Medical Genetics
	Individual	1	2	3	4	5	6	7	8
	Gene	GRIN1	GRIN1	GRIN1	GRIN1	GRIN2B	GRIN2B	GRIN2B	GRIN1
	Mutation	c.1658C>T, p.(Ser553Leu)	c.1972G>T, p.(Asp658Tyr)	c.1957G>A, p.(Ala653Thr)	c.2231G>A, p.(Cys744Tyr)	c.2437C>G, p.(Leu813Val)	c.2438T>C, p.(Leu813Pro)	c.2453T>C; p.(Met818Thr)	c.1422C>A, p.(Tyr474*)
	Inheritance	De novo	De novo	De novo	De novo	De novo	De novo	De novo	Homozygous, inherited
	Age at last review	19 m	n/a	25 GW	30 GW	23 m	2.5 y	15 m	6 d
	Sex	Male	Male	Male	n/a	Male	Male	Male	Female
	DD	Severe	Severe	n/a	n/a	Severe	Severe	Severe	n/a
	Age sz onset	Birth/prenatal	4 m	n/a	n/a	4 m	2 y 3 m	10 w	Early postnatal
	Sz type	n/a	Absences, drops, tonic extensions	n/a	n/a	Epileptic spasms, tonic sz	GTCS with fever	Infantile movements spasms, rapid and rolling eye	Status epilepticus
	Refractory sz	No	Yes	n/a	n/a	Yes	No	No	Yes
							Spastic
							quadriplegia
							Truncal
	Neurology	Hypertonia	Spastic quadriplegia	n/a	n/a	Spastic quadriplegia	hypotonia and limb	Hypertonia	n/a
							hypertonia,
							pseudobulbar
							palsy
	Dysmorphism	Long palpebral fissures, shallow orbits, fingers overlapping	Thin upper lips, anteverted bridge wide nasal nostrils, high frontal hairline,	Prominent philtrum, nostrils anteverted broad nasal bridge,	Marked nasal ridge, camptodactyly face, prominent philtrum, flat	No	No	No	No
				https://mc.manuscriptcentral.com/jmedgenet

Abbreviations: Age, y(ears), m(onths), w(eeks), d(ays); ACC; agenesis of corpus callosum, A>P, anterior more severe than posterior; BG, basal ganglia; BS, brainstem; CC, corpus callosum; GW, gestational weeks; GTCS, generalized tonic-clonic seizures; LV, lateral ventricles; n/a, not applicable; n/k, not known; SD, standard deviations above or below mean; sz, seizures; WM, white matter https://mc.manuscriptcentral.com/jmedgenet

Neuropathological findings in GRIN1 patients

Autopsy and neuropathological examination were performed in individuals 3, 4 and 8.

  We presented seven new patients and reviewed all previously reported NMDAR MCD patients with de novo heterozygous variants in either GRIN1 or GRIN2B. In addition, we provided the first reports of neuropathological findings in two individuals with heterozygous and one individual with homozygous variants in GRIN1. Our findings expand the current understanding of the phenotypic spectrum of NMDAR-associated MCD and provide insight into the suspected underlying pathophysiological mechanisms. tubulinopathies and the spectrum observed in individuals with variants in GRIN1 or GRIN2B, tubulinopathies are often associated with abnormalities of the corpus callosum, brainstem and cerebellar hypo-or dysplasia, possibly due to the role microtubules play in neuronal migration and axon development.[24] In contrast, the cerebellum and brainstem were usually normal in individuals with variants in either GRIN1 or GRIN2B. Prior to genetic testing, variants in GRIN1 and GRIN2B were not suspected based on clinical or imaging features in the individuals presented in this cohort. This might be due to the small number of individuals with NMDAR-related MCD reported previously. During foetal life, the discovery of in patients with and without MCD and variants in NMDAR.[START_REF] Ohba | GRIN1 mutations cause encephalopathy with infantile-onset epilepsy, and hyperkinetic and stereotyped movement disorders[END_REF][START_REF] Lemke | Delineating the GRIN1 phenotypic spectrum: A distinct genetic NMDA receptor encephalopathy[END_REF][START_REF] Fry | De novo mutations in GRIN1 cause extensive bilateral polymicrogyria[END_REF][START_REF] Nishimura | Arthrogryposis multiplex congenita with polymicrogyria and infantile encephalopathy caused by a novel GRIN1 variant[END_REF][START_REF] Platzer | GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects[END_REF] Interestingly, none of the individuals in our series have been reported to have abnormal movements. However, it is possible that abnormal movements were present but not recognized, limited by limb spasticity, or misdiagnosed as seizures.We provided the first neuropathological reports of three individuals with variants in GRIN1. Individuals 3 and 4 were reported with dysgyria resembling polymicrogyria on brain MRI and de novo heterozygous pathogenic variants in GRIN1. Neuropathological examination confirmed polymicrogyria. Both individuals also had extreme microcephaly, nodular heterotopias in the periventricular and intermediate zones, gaps of the glia limitans and overmigration foci in the meninges, arhinencephaly, dysplastic basal ganglia and Noteworthy, using NMDAR RNA interference with in utero electroporation in the cortex of rat embryos, Jaing et al.[START_REF] Jiang | The GluN2B subunit of N-methy-D-asparate receptor regulates the radial migration of cortical neurons in vivo[END_REF] found severely delayed radial migration, with more than 80% of migrating MAP2-positive projection neurons which could not reach the upper layers of the cortical plate and with 30% of the cells remaining located in the intermediate zone. In addition, it has been shown that developmental disruption of NMDA receptors has major effects on interneuron localization leading to inhibitory

	Journal of Medical Genetics Journal of Medical Genetics Journal of Medical Genetics	Page 22 of 53
	DISCUSSION network deficits, in so far as ionotropic NMDARs are normally expressed in tangentially
	migrating interneurons.[27]
	It remains uncertain why a subset of patients with NMDAR variants develops severe
	MCDs. It has already been highlighted that GRIN-variants associated with diffuse dysgyria
	C o n f i d e n C o n f i d e n C o n f i d e n tend to have gain of function (GOF) effects, as has been shown by functional studies for two C o variants in GRIN1 (p.(Ala653Thr) and p.(Cys744Thr)) and one variant in GRIN2B n n e d i f (p.Met818Thr) presented here, while many non-MCD variants are hypofunctional.[9, 13]
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	the information is available have microcephaly (ranging from -2.0 to -7.1 SD in GRIN1
	patients). Six out of 23 (26%) of non-MCD patients with GRIN1 variants reported by Lemke
	21
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(Figure

4I-L

). These findings highly suggest that functional NMDARs are essential for proper radial and tangential migration without which neurons cannot reach their final location.

For the 25 patients with heterozygous variants in either GRIN1 or GRIN2B that have now been reported in this series and in the wider literature, we found a striking similarity in imaging and phenotypic features. The predominant imaging pattern of MCD in both groups was an extensive diffuse, bilateral dysgyria resembling polymicrogyria. Commonly associated brain malformations in both groups include enlarged ventricles, hippocampal dysplasia, hypoplastic corpus callosum, and enlarged or dysmorphic basal ganglia. Hippocampal dysplasia has been reported on MRI in 5/15 individuals with variants in GRIN1 and in 5/9 patients with variants in GRIN2B. This feature was confirmed by neuropathological examination in individual 4 and in an individual with a homozygous GRIN1 variant (individual 8) in our series. The hippocampal abnormalities are likely due to the high expression of NMDAR in the hippocampus.

[23]

Abnormal basal ganglia were found in 6/14 and 8/9 of individuals with variants in GRIN1 and GRIN2B, respectively, likely reflecting the importance of glutaminergic signaling passing through the basal ganglia. Both hippocampal dysplasia and dysmorphic basal ganglia are rare in patients with MCD in general. Individuals with variants in tubulin genes, referred to as tubulinopathies, also show complex brain malformations, including dysgyria and dysmorphic basal ganglia. Rarely, hippocampal hypoor dysplasia is present, most frequently in patients with variants in TUBA1A.

[24, 25]

Although there appears to be significant overlap of clinical and imaging findings between Page 20 of 53 https://mc.manuscriptcentral.com/jmedgenet microcephaly or microlissencephaly with or without corpus callosum agenesis may correspond to a constellation of etiologies, either environmental or genetic and there are no specific signs identified to date which could point to GRIN1 and GRIN2B as candidate genes. The homogeneous imaging spectrum observed in individuals with GRIN1 and GRIN2Bassociated MCD, including diffuse dysgyria, dysmorphic basal ganglia and hippocampal dysplasia without any infratentorial lesions may therefore be a potential diagnostic clue and should trigger consideration of pathogenic variants in NMDAR based on imaging findings in the future. Clinical features included severe or profound developmental delay or intellectual disability, early onset epilepsy, microcephaly, spastic quadriparesis, and cortical visual impairment. Microcephaly was more common in NMDAR-MCD patients than in NMDARpatients without MCD, and was present in all seven new patients presented here. Overall, et al. [9] and 7/56 (12.5%) non-MCD patients with GRIN2B missense variants reported by Platzer et al.[15] had microcephaly. Hyperkinetic and stereotyped movement disorders that Page 21 of 53 https://mc.manuscriptcentral.com/jmedgenet 20 have been described hippocampi as well as cerebellar hypoplasia. In addition, we provide first evidence that homozygous loss of function (LOF) variants also cause MCD. Neuropathological examination of individual 8 carrying homozygous GRIN1 variants has revealed microcephaly and a SGP associated with defective lamination of the cortex and malrotated hippocampi. Taken together, the features observed argue for a crucial role of GRIN1 in the morphological organization of the different neuroanatomical structures during fetal life.

Immunohistochemical findings also strongly argue for decreased neuron production as well as delayed and inappropriate radial and tangential migration resulting in microcephaly with polymicrogyria or simplified pattern.

  24for the first time provide evidence that GRIN1 is implicated in the generation, migration and cortical positioning of neurons and interneurons in humans, ultimately leading to MCD.

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Table 4 . ACMG-AMP criteria used to classify the NMDAR variants.

 4

	Journal of Medical Genetics

manuscriptcentral.com/jmedgenet https://mc.manuscriptcentral.com/jmedgenet

Table 5 . In silico predictions for the NMDAR missense variants and the homozygous GRIN1 variants

 5

	Gene	GRIN1	GRIN1	GRIN1	GRIN1	GRIN2B	GRIN2B	GRIN2B	GRIN1
	Mutation	c.1658C>	c.1972G>	c.1957G>	c.2231G>	c.2437C>	c.2438T>	c.2453T>	c.1422C>
	(NM_007327.	T,	T,	A,	A,	G,	C,	C,	A,
	3,	p.S553L	p.D658Y	p.A653T	p.C744Y	p.L813V	p.L813P	p.M818T	p.Y474*
	NM_000834.
	3)
	Genomic	chr9:g.140	chr9:g.140	chr9:1400	chr9:g.140	chr12:g.13	chr12:g.13	chr12:g.13	chr9:1400
	position	056649C>	057150G>	57135G>	057680G>	720120G>	720119A>	720104A>	55823C>
	(hg19)	T	T	A	A	C	G	G	A
	SIFT	0.001	0	0.001	0	-	-	-	-
		(Damagin	(Damagin	(Damagin	(Damagin
		g)	g)	g)	g)
	Polyphen2	1	1	1	1	1	1	0.999	-
	(HDIV)	(Probably	(Probably	(Probably	(Probably	(Probably	(Probably	(Probably
		damaging)	damaging)	damaging)	damaging)	damaging)	damaging)	damaging)
	Polyphen2	1	1	1	1	0.999	1	0.999	-
	(HVAR)	(Probably	(Probably	(Probably	(Probably	(Probably	(Probably	(Probably
		damaging)	damaging)	damaging)	damaging)	damaging)	damaging)	damaging)
	LRT	0	0	0	0	0	0	0	0
		(Unknown	(Deleterio	(Deleterio	(Deleterio	(Deleterio	(Deleterio	(Deleterio	(Unknown
		)	us)	us)	us)	us)	us)	us)	)
	MutationTast	1 (Disease	1 (Disease	1 (Disease	1 (Disease	1 (Disease	1 (Disease	1 (Disease	-
	er	causing)	causing)	causing)	causing)	causing)	causing)	causing)
	MutationAss	3.415	2.645	3.21	3.91	3.085	3.23	2.395	-
	essor	(Medium)	(Medium)	(Medium)	(High)	(Medium)	(Medium)	(Medium)
	FATHMM	2.01	0.55	-0.32	0.51	-	-	-	-
		(Tolerable	(Tolerable	(Tolerable	(Tolerable
		)	)	)	)
	PROVEAN	-5.03	-6	-3.35	-9.82	-	-	-	-
		(Damagin	(Damagin	(Damagin	(Damagin
		g)	g)	g)	g)
	VEST3	0.899	0.574	0.749	0.99	0.851	0.981	0.935	-
		(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin
		g)	g)	g)	g)	g)	g)	g)
	MetaSVM	-0.447	-0.328	0.018	0.227	0.106	0.257	-0.36	-
		(Tolerable	(Tolerable	(Damagin	(Damagin	(Damagin	(Damagin	(Tolerable
		)	)	g)	g)	g)	g)	)
	MetaLR	0.202	0.34	0.486	0.473	0.503	0.547	0.329	-
		(Tolerable	(Tolerable	(Tolerable	(Tolerable	(Damagin	(Damagin	(Tolerable
		)	)	)	)	g)	g)	)
	M_CAP	0.425	0.914	0.823	0.48	0.606	0.919	0.551	-
		(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin
		g)	g)	g)	g)	g)	g)	g)
	CADD	32	26.7	28.1	28.4	25.4	28.2	26	36
		(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin
		g)	g)	g)	g)	g)	g)	g)	g)
	DANN	0.999	0.995	0.999	0.997	0.998	0.999	0.994	0.995
		(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin
		g)	g)	g)	g)	g)	g)	g)	g)
	FATHMM_	0.937	0.96	0.944	0.955	0.974	0.985	0.985	0.283
	MKL	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Tolerable
		g)	g)	g)	g)	g)	g)	g)	)
	Eigen	0.578	0.604	0.623	0.874	0.638	0.868	0.807	-0.385
		(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Tolerable
		g)	g)	g)	g)	g)	g)	g)	)
	GenoCanyon	1	1	1	1	1	1	1	1
		(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin	(Damagin
		g)	g)	g)	g)	g)	g)	g)	g)
	fitCons	0.598	0.658	0.658	0.652	0.554	0.554	0.554	0.598
		(Tolerable	(Tolerable	(Tolerable	(Tolerable	(Tolerable	(Tolerable	(Tolerable	(Tolerable
		)	)	)	)	)	)	)	)

Functional evaluation of a human glutamate receptor variant p.Cys744Tyr in GRIN1 in vitro a. METHODS cDNA

  Plasmid Constructs: The p.Cys744Tyr and the p.Ala653Thr GRIN1 variants were introduced into a GluN1 cDNA (RefSeq NM.007327.3) plasmid construct , and the p.Met818Thr GRIN2B variant into the GluN2B cDNA (RefSeq NM_000834.3) via a Quikchange (Agilent) mutagenesis protocol. All experiments used cDNAs encoding human gene sequences verified with Sanger sequencing (Eurofins). mRNA was synthesized from cDNA using mMESSAGE mMACHINE (Invitrogen). The oocyte membrane potential was clamped at -40 mV. After a steady baseline was obtained, oocytes were perfused with increasing concentrations of L-glutamate (seven concentrations selected from 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 M) for 0.75 min duration each in the continuous presence of 100 M glycine. Results at each L-glutamate concentration were normalized to the maximum receptor activation levels (defined as 100%) and the EC50 values obtained by fitting concentration-response data with Equation 1 (below).

	mRNA Injections: The cRNAs for GluN1-C744Y, GluN1-A653T or GluN1-WT were combined,
	separately, with human GluN2A-WT cRNA, or GluN1-WT and GluN2B-M818T were combined
	separately, and injected into stage V and VI oocytes and incubated at 16 C for 2-7 days in Barth's
	culture medium containing (in mM): 88 NaCl, 2.4 NaHCO3, 1 KCl, 0.33 Ca(NO3)2, 0.41 CaCl2, 0.82
	MgSO4, and 10 HEPES (pH 7.4). For experiments, oocytes were removed from the incubator and
	perfused in recording Barth's solution for two-electrode voltage clamp (TEVC) electrophysiological
	recordings. Conditions for each experiment are below.
	L-Glutamate dose response studies: The recording solution for these studies contained (in mM): NaCl
	(90), KCl (1.0), BaCl2 (0.5), HEPES (10), and EDTA (0.01), adjusted to pH 7.4 (at 23 degrees C) with
	NaOH.

  The oocyte membrane potential was clamped at -60 mV. After a steady baseline was obtained, oocytes were maximally activated with 100 M L-glutamate and 100 M glycine and were then perfused with increasing concentrations of Mg 2+ (3, 10, 30, 100, 300, and 1000 M) in the presence of maximal glutamate and glycine. The current responses to glutamate and glycine at each Mg 2+ concentration were normalized to the maximum receptor activation levels in the absence of Mg 2+ (defined as 100%) and the IC50 values obtained by fitting concentration-inhibition data with Equation 2 (below).Zn 2+ dose inhibition studies: Oocytes expressing recombinant human glutamate receptors were perfused with recording solution containing (in mM): NaCl (90), KCl (1.0), BaCl2 (0.5), Tricine[START_REF] Xiangwei | De Novo Mutations and Rare Variants Occurring in NMDA Receptors[END_REF], and HEPES[START_REF] Xiangwei | De Novo Mutations and Rare Variants Occurring in NMDA Receptors[END_REF] adjusted to pH 7.3 (at 23 degrees C) with NaOH. The oocyte membrane potential was clamped at -20 mV. After a steady baseline is obtained, oocytes were maximally activated with 50 M L-glutamate and 50 M glycine, and then in the continuous presence of maximal glutamate and glycine, were perfused with increasing concentrations of Zn 2+ (1, 3, 10, 30, 100, and 300 nM). Results at each Zn 2+ concentration are normalized to the maximum receptor activation levels without Zn 2+ (defined as 100%) and IC50 values obtained by fitting concentration-inhibition data with Equation2(below).

	1, 0.3, 1, 3, 10, 30 and 100 M) for 0.75 min duration each in the continuous presence of 100 M L-glutamate. Results at each glycine concentration were normalized to the maximum receptor activation levels (defined as 100%) and the EC50 values obtained by fitting concentration-response data with Equation 1 (below). Mg 2+ dose inhibition studies: The recording solution for Mg 2+ studies contained (in mM): NaCl (90), KCl (1.0), BaCl2 (0.5), and HEPES (10) adjusted to pH 7.4 (at 23 degrees C) with NaOH. pH studies: The recording solution for these studies contained (in mM): NaCl (90), KCl (1.0), BaCl2 (0.5), HEPES (10), and EDTA (0.01), adjusted to either pH 6.8 or pH 7.6 (at 23 degrees C) with HCl or NaOH. The oocyte membrane potential was clamped at -40 mV. After a steady baseline was obtained o n f i d e n t i a l : F o r R e v i e w O n l in pH 7.C y

[START_REF] Hamdan | Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability[END_REF]

recording buffer the oocytes were maximally activated with 100 M L-glutamate and 100 M glycine in pH 7.6 buffer. Following a washout period to reestablish baseline, the oocytes were then maximally activated with 100 M L-glutamate and 100 M glycine in pH 6.8 buffer. The percent current at pH 6.8 was then determined compared to the current at pH 7.6 (defined as 100%).

  was performed in the two foetuses (individuals 3 and 4) and one neonate (individual 8) following standardized protocols. Fetal biometric data were evaluated according to Guilhard-Costa et al.(2002). The brains were fixed in a 10%

	Journal of Medical Genetics
	4. Neuropathological and immunohistochemical studies
	a. Morphological studies
	A complete autopsy
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	y y
	11

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Table 7 :

 7 comparative semi-quantitative analysis of immunolabelling in the germinative and intermediate zones as well as in the different layers of the frontal cortex of affected and control patients

				Journal of Medical Genetics		Page 54 of 53
	Antibodies	Foetus 1	Control	Foetus 2	Control	Patient 3	Control
		25 WG	25 WG	30 WG	30 WG	38 WG, dead at	39 WG
						postnatal day 6
	PAX6	++ LGE	+++ LGE	0	0	0	0
	SOX2	++ LGE	+++ LGE	++ LGE	+++ SVZ	NA	++ VZ
				+ SVZ	+++ LGE
	Ki67	+ LGE	++ LGE	+++ LGE	++ LGE	0	0
				+ SVZ	+++ OSVZ
	GABA	0 LGE	+++ LGE	+ LGE and SVZ	++ diffuse CP	+ in layer II and III	++ all layers
		+ IZ	+++ VZ, SVZ,	+ Superficial
		+ under the CP	++ CP	micropolygyric CP
	Calretinin	0 LGE and VZ	+ IZ	+ LGE	+ VZ	Fibre network layer I	++ layers II and III
		+ Layer I	+ VZ	+ superficial	+ LGE	+ interneurons layer II
		(Cajal Retzius cells)	+ LGE	micropolygyric CP	++ Layers II, III and
		0 in micropolygyric CP		+ IZ	V
		Dispersed in nodules		+++ nodules
	MAP2	Periventricular nodules	+++ VZ	+ micropolygyric CP	++ layer II and III	+ Immature neurons	++ layers III and V
		+ Migrating neurons in IZ	Layers III and V	Dysmorphic neurons	+++ layer V
		0 CP
	SATB2	Immature neurons in	+ VZ and LGE	0	Layers II-IV	0	Layers II-IV,
		layer II	++ IZ		+++ in layer II		+++ in layer II
			+++ diffuse CP
	CTIP2	0	+++ VZ and LGE	0	++ layer V	0	++ layers V and VI
			+++ prospective layer
			V
	WG: weeks of gestation; VZ: germinative zone of the dorsal telencephalon; SVZ subventricular zone; LGE: lateral ganglionic eminences; IZ: intermediate zone; CP: cortical
	plate
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https://mc.manuscriptcentral.com/jmedgenet Journal of Medical Genetics

Journal of Medical Genetics

Acknowledgments

The authors would like to thank the patients, families and scientists who contributed to this work, and Rebecca Bui and Dan Teuscher for technical assistance. SB, AL, KS, GM, UH, AJ, AEF and NBB are members of the COST Action CA16118 Neuro-MIG.

Funding: The DDD study presents independent research commissioned by the Health Innovation Challenge Fund [grant number HICF-1009-003]. This study makes use of DECIPHER (http://decipher.sanger.ac.uk), which is funded by Wellcome. See Nature PMID: 25533962 or www.ddduk.org/access.html for full acknowledgement. AJ was funded by an FWO Senior Clinical Investigator Fellowship. SB received funding from the Scientific Fund Willy Gepts. DB is supported by NIHR Research Professorship RP-2016-07-011. SFT received funding from the NIH-NINDS (NS111619). HY is supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health (R01HD082373) and the National Institute of Mental Health (MH127404). SJM is supported by a grant from the National Institute on Aging (R21AG072142). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. S.F.T. is principal investigator on research grants from Biogen and Janssen to Emory; is a member of the Scientific Advisory Board for Eumentis Inc, Sage Therapeutics, the GRIN2B Foundation, the

Competing Interests:

No conflict of interest to declare.

CureGRIN Foundation; is cofounder of NeurOp Inc and Agrithera Inc., and is co-inventor on Emory-owned Intellectual Property.

Ethics declaration

Informed consent was obtained from the legal guardians of the patients described in this study. The study was approved by the ethical committee of the UZ Brussel (B.U.N.143201214360). The DDD study was approved by the Cambridge South Research Ethics Committee (10/H0305/83).

Contributorship Statement

A.E.F., N.B-B. and A.J. conceived the work. SB, A.L., A.E.F., S.J.M. and N.B-B assisted with data acquisition. SB, A.L and A.E.F drafted the manuscript, which was revised and approved by all.


Sylvain Charvet

Maurice Médebielle

Julien C Vantourout

Mn-Mediated α-Radical Addition of Carbonyls to Olefins: Systematic Study, Scope, and Electrocatalysis

A systematic study of the manganese-mediated α-radical addition of carbonyl groups to olefins is presented. After an in-depth investigation of the parameters that govern the reaction, a first round of optimization allowed the development of a unified stoichiometric set of conditions, which were subsequently assessed during the exploration of the scope. Due to observed limitations, the knowledge accumulated during the initial study was reengaged to quickly optimize promising substrates that were so far inaccessible under previously reported conditions. Altogether these results led to the creation of a predictive model based on the pKa of the carbonyl compound and both the substitution and geometry of the alkene coupling partner. Finally, a departure from the use of stoichiometric manganese was enabled through the development of a robust and practical electrocatalytic version of the reaction.

INTRODUCTION

One of the central challenges of our century is the development of sustainable chemical processes. 1 Following this concept, the use of first row transition metals has become essential in catalysis. 2 Manganese is abundant, inexpensive, and nontoxic and participates in a myriad of transformations due to the wide range of oxidation states it can adopt (from 0 to +7, [Ar]3d 5 4s 2 ). 3 In the framework of a new research program dedicated to manganese catalysis, we are particularly interested in the systematic study of reactions previously neglected due to low practicality and lack of mechanistic understanding.

For our first study, we selected the field of α-alkylation of carbonyls as it is a central and well-established transformation in organic synthesis that could still benefit from improvement f1 (Figure 1). [START_REF] Lamoureux | C. A Comparison of Several Modern Alkylating Agents[END_REF] Indeed, the coupling of enolates with nucleofugecontaining molecules under basic conditions is extensively employed, but its low atom-economy makes it often industrially impracticable. [START_REF] Clayden | J. First Asymmetric Total Synthesis of (+)-Sparteine[END_REF] Similarly, modern procedures employing a combination of a metal-based catalyst with directing amines have allowed the use of alkenes as coupling partners, which circumvent any waste generation. [START_REF] Nakamura | α-Alkylation of Ketones by Addition of Zinc Enamides to Unactivated Olefins[END_REF] However, the expensive catalysts and/or reagents employed in these protocols make them difficult to implement on scale. Despite the current limitations associated with the Mn-mediated 69 aldehydes, and carboxylic acids. 11 In addition, the possibility 70 for the carbonyl moieties to react with nonactivated olefins 71 using earth abundant manganese renders this transformation 72 highly attractive and broadly applicable, especially in the 73 context of natural product synthesis (Scheme 1A.4). 14 While 74 the Snider group among others reported several intramolecular 75 polycyclization cascades, 15 the Mamdapur groups applied the 76 intermolecular version of the Mn-mediated α-radical addition 77 of carbonyl groups to olefins as a key step for the synthesis of 78 queen bee pheromone 9 and himasecolone 10, respectively. 16 79 Despite all the advantages it offers, the intermolecular version 80 of the reaction still suffers from major limitations such as (1) 81 the absence of a general set of conditions; (2) the need for a 82 large amount of the carbonyl starting material to be employed 83 (usually more than 10 equiv); (3) the use of acetic acid as the 84 solvent which lowers the functional group tolerance; and (4) 85 the lack of understanding with regard to some reaction 86 parameters including temperature, concentration, atmosphere, 87 and more importantly, the nature of the alkene coupling 88 partner (Scheme 1A.4). These limitations hamper the 89 widespread use of this powerful transformation. Another 90 significant drawback that reduces the broad application of 91 this reaction is the (over)stoichiometric amount of the 92 Mn(OAc) 3 catalyst often required to afford satisfactory 93 yields. 11 Such an excess amount of the manganese catalyst 94 leads to a tedious workup, as exemplified in several studies. 15e 95 To overcome this issue, several groups have attempted to run 96 the reaction under oxidative conditions to regenerate Mn(III) 97 from Mn(II). 11,17 As a selected example, the Ishii group 98 reported the coupling between cyclohexanone 3 and octene 11 99 to afford desired product 12 in 59% yield (Scheme 1A.5). 18 100 This set of conditions only requires 0.1 mol % of the 101 Mn(OAc) 2 catalyst and 0.05 mol % of the Co(OAc) 2 co-102 catalyst to be employed under a mixed atmosphere of oxygen 103 and nitrogen. However, the large amount of the ketone starting 104 material and the use of AcOH as the solvent remain two major 105 limitations which reduce the applicability of the reaction. As an 106 alternative to chemical oxidative conditions, it was demon-107 strated that Mn(II) could be oxidized to Mn(III) under 108 electrochemical conditions (Scheme 1A.5). [START_REF] Shundo | Selective Coupling of Non-Activated Olefins with Ethyl Cyanoacetate by Mn 3+ -Mediated Anodic Oxidation[END_REF] In these studies, 109 the products are obtained in good yield using low Mn(II) 110 catalyst loadings (down to 10 mol %). Nevertheless, the use of 111 solvent quantities of AcOH, the necessity to run the reaction in 112 a divided cell, and the absence of a general procedure reduce 113 the synthetic value of this catalytic approach.

114

The aforementioned limitations have certainly lowered the 115 uptake of the Mn-mediated α-radical addition of carbonyl 116 groups to olefins, 11f and although this reaction has been 117 exemplified multiple times in the literature, it has only been 118 sporadically studied. 20 We recognized that if rendered 119 practical, such a reaction could undoubtedly impact the field 120 of carbonyl α-functionalization. Therefore, we decided to (1) 121 investigate the parameters that govern this transformation; (2) 122 assemble a general set of stoichiometric reaction conditions; 123 (3) evaluate the scope and its limitations; (4) reengage the 124 knowledge accumulated during the initial study to quickly 125 optimize low yielding substrates; and (5) depart from the use 126 of stoichiometric manganese with the development of a general 127 electrocatalytic version of the reaction (Scheme 1B).

RESULTS AND DISCUSSION

128 3.1. Systematic Study of the Reaction Parameters. We 129 began our investigation by studying the parameters that govern the Mn-mediated α-radical addition of carbonyls to olefins s2 between cyclohexanone 3 and 1-hexadecene 13 (Scheme 2A).

As most of the existing intermolecular processes suffer from the requirement for a large amount of the carbonyl compound to be used under oxidative conditions, the excess of ketone 3 was limited to 3 equiv and the reaction was run under argon.

Based on previously reported conditions, Mn(OAc) 3 was selected as the stoichiometric catalyst source and the reaction was conducted at 70 °C. Inspired by the elegant Mn(III)catalyzed intramolecular cyclization study disclosed by the Snider group, where the authors described the impact of the solvent and especially the beneficial effect of using EtOH to enhance the HAT termination step, we initially studied this parameter. 21 To our surprise, AcOH, which is the preferred solvent for the majority of previously reported conditions, only afforded 14% of desired product 14 (Scheme 2A.1). This is potentially due to the limited amount of ketone (3 equiv) used in the reaction, which cannot compete with the competitive lactone formation reported by Finkbeiner and Heiba (Scheme 1A.1). DMA, DME, DMF, MeCN, DCE, and HFIP were also tested but only delivered moderate yields (22-46%, Scheme 2A.1). This trend can be rationalized by the reduced ability of these solvents to facilitate the final HAT process as the H-atom source, constraining the ketone starting material or the desired product to serve this role. The subsequent radicals can then result in the formation of unwanted byproducts (see the Supporting Information for details). As expected, based on Snider initial disclosure, out of all the solvents that were screened, EtOH conferred the best result (74% yield), while toluene provided a moderate yield of 58% (Scheme 2A.1). 21 This result matches Snider initial observation. In each case, the corresponding stabilized α-hydroxy or benzylic radical afforded by the HAT termination step can explain why these solvents offered an improved outcome. The remarkably enhanced conversion observed in EtOH can also be supported by several studies which previously highlighted its ability to enhance the reactivity of manganese-based processes. 22 Screening of commercially available catalyst sources was then attempted using EtOH as the solvent (Scheme 2A.2). Unfortunately, Mn(dpm) 3 and Mn(acac) 3 resulted in none and 14% conversion to product 14, respectively. The steric hindrance engendered by dpm or acac ligands surrounding the metal center obstructs the formation of coordinated intermediate 4, which impedes product formation. In addition, such ligands could lead to the formation of undesired α-functionalized byproducts. A non-commercially available Mn(pic) 3 catalyst was also tried under the reaction conditions but did not provide a satisfactory yield compared to Mn(OAc) 3 (see the Supporting Information for details). Unsurprisingly, the use of Mn(OAc) 2 did not afford any product as oxidation to Mn(III) is impossible under an argon atmosphere. The influence of temperature on the reaction was also investigated (Scheme 2A.3). Temperatures lower than 60 °C considerably diminished the yield as it decreased the solubility of the manganese catalyst, while temperatures higher than 70 °C significantly increased byproduct formation. The impact of the amount of manganese was then studied (Scheme 2A.4). While increasing the number of equivalents from 1 to 1.5 improved the yield of compound 14 from 70 to 74%, lowering the loading from 1 to 0.5 equivalents resulted in a decreased yield of 51%. This result is in accordance with the proposed mechanism where Mn(III) is reduced to Mn(II) to generate keto-radical 6. Under inert conditions, Mn(II) cannot be re-

The Journal of Organic Chemistry D 193 oxidized and a (super)stoichiometric amount of manganese 194 needs to be employed. Of note, using a large amount of the 195 manganese catalyst (e.g., 3 equiv) resulted in low conversion to 196 the desired product (44%), possibly due to precipitation of the 197 catalyst. Several additives were screened, and their impact on the reaction efficiency was studied (Scheme 2A. The Journal of Organic Chemistry 203 step did not increase the yield of the reaction and instead 204 considerably hampered conversion. We next turned our 205 attention to the atmosphere under which the coupling reaction 206 was being conducted. Running the reaction under argon 207 provided 70% of desired product 14, while an atmosphere of 208 oxygen only afforded 44% (Scheme 2A.6). A careful analysis of 209 the byproduct distribution allowed us to explain the drastic 210 differences observed between an inert and oxidizing environ-211 ment (see the Supporting Information for details). Indeed, 212 under air or oxygen, the ketone starting material is consumed 213 by undesired processes leading to dimeric and α-oxidized 214 species, thus lowering the overall yield of the reaction. Based 215 on the elegant study from the group of Ishii, the impact of 216 using a Co(II) co-catalyst was also reevaluated (vide supra). [START_REF] Kagayama | A Remarkable Effect of Bases on the Catalytic Radical Addition of Cyanoacetates to Alkenes Using a Mn(II)/Co(II)/O 2 Redox System[END_REF] 217 Attempts to apply these conditions to our system resulted in 218 low efficiency and only gave a 21% yield of desired product 14. 219 A reduction in ketone stoichiometry (1 or 2 equiv) diminished 220 the yield considerably, and compound 14 was only obtained in 221 27 and 34% yields, respectively (Scheme 2A.7). The use of a 222 large amount of ketone (5 or 10 equiv) delivered high yields, 223 matching with previously reported results from the literature. 11 224 Finally, as both the solubility of the manganese catalyst and 225 overall concentration of the ketone starting material appeared 226 to greatly impact the yield of the reaction, we hypothesized 227 that the concentration should also play an important role in the 228 outcome of the transformation (Scheme 2A.8). Moving from a 229 concentration of 0.5 to 0.1 M significantly decreased the yield 230 from 70 to 38%. Similarly, the yield of the reaction was 231 markedly lowered when the concentration was too high due to 232 catalyst insolubility and lower stirring efficiency. 233 3.2. Optimized Stoichiometric Set of Reaction 234 Conditions. Using the knowledge gained throughout this 235 in-depth study, a simple and general Mn-mediated α-radical 236 addition of carbonyls to olefins was developed (see the 237 Supporting Information for details). Running the reaction with 238 a slight amount of Mn(OAc) 3 in EtOH at suitable temperature 239 (70 °C) and concentration (0.75 M) under an atmosphere of 240 argon delivered desired product 14 in 67% isolated yield with 241 only 3 equiv of the carbonyl starting material (Scheme 2B). 242 3.3. Scope of the Reaction. With the optimized 243 conditions in hand, we first started to assess the carbonyl s3 244 scope (Scheme 3A). Ketones such as cyclopentanone, acetone, 245 and indanone delivered compounds 15, 16, and 17 in 70, 59, 246 and 40% isolated yields, respectively. Noteworthily, the 247 conversion for compounds 16 and 17 (83 and 97%, 248 respectively) was much higher than the isolated yield due to 249 challenging purification. α-Tetralone, which was known to be 250 unreactive under previously reported conditions, afforded 251 compound 18 in 17% yield. Boc-protected piperidin-4-one 252 afforded desired product 19 in 63% isolated yield. Strikingly, 253 this compound was only obtained in a trace amount following 254 the original literature conditions, which highlights the 255 importance of using EtOH instead of AcOH to improve the 256 functional group tolerance. Nitroacetate did not give the 257 desired linear product but the cyclized compound 20 in a 258 moderate isolated yield of 33%. Other carbonyls including 259 diester, keto-ester, and cyanoacetate proved compatible under 260 the reaction conditions with compounds 21, 22, and 23 261 obtained in good yields (63-74%). Unfortunately, due to 262 higher α-C-H pKa values, amides and esters did not react 263 under the reaction conditions (compounds 32 and 33, Scheme 264 3C). Similarly, compound 34 issued from the coupling between methyl isopropyl ketone (MIPK) and 1-hexadecene 13 was only detected in negligible amounts (traces in GCMS).

Regarding the alkene scope, stabilized disubstituted olefins such as prop-1-en-2-yl acetate when reacting with cyclohexanone 3 delivered product 27 in 61% isolated yield.

Alkynes were also successfully coupled under the reaction conditions with alkene 29 obtained in 43% yield (14:1 E/Z ratio). Functionalization of valuable alkenes such as camphene and (+)-β-citronellene afforded different outcomes with compounds 30 obtained in 66% yield, while only a trace amount of compound 31 was detected. In addition, cyclohexene when coupled with indanone delivered compound 28 in 16% isolated yield, highlighting that 1,2-disubstituted nonstabilized olefins are challenging compounds to functionalize. This trend was further confirmed when 1-methylcyclohex-1-ene, (E)-non-4-ene, and (Z)-non-4-ene were used as alkene coupling partners with no desired or trace product formation observed.

Regarding functional group tolerance, the optimized reaction conditions proved highly efficient. Indeed, ester (21-23 and 27), cyano (23), nitro (20), free (25), or TBS (24)-protected alcohols, and acetyl groups (27) were all tolerated under the reaction conditions and afforded the desired products from moderate to good yields (27-74%).

Notably, halogen moieties were also stable under the reaction conditions with bromine containing compound 25 obtained in 56% yield. To further assess the functional group tolerance, a robustness test was performed by adding 1 equiv of an additive bearing a specific reactive handle (Scheme 3D). [START_REF] Collins | A Robustness Screen for the Rapid Assessment of Chemical Reactions[END_REF] While phenol, amide, and carboxylic acid moieties did not impact the outcome of the reaction, amine-and nitro-based compounds hampered both the conversion and yield. Interestingly, pyridine lowered the conversion (73% instead of 92%) but afforded a serviceable yield (53% instead of 74%).

All these results can be rationalized based on the nature of the olefin or carbonyl coupling partners. Alkenes that afford secondary radicals are not oxidized by Mn(III) allowing the HAT step to proceed efficiently. However, when they react with highly reactive carbonyl, the secondary radical can engage with the oxygen and then undergo oxidation to give lactones or dihydrofurans such as compound 20. 1,2-Disubstituted alkenes work poorly since the initial addition is slow. Finally, 1,1disubstituted alkenes and styrenes are the most reactive alkenes, but the radicals issued from the initial addition step are subsequently oxidized, so there is little or no HAT product as exemplified by compound 30.

Overall, the optimized set of reaction conditions delivered a broad scope, but some unexpected and contrasting results highlighted the difficulty to identify a truly general set of conditions, especially with respect to the alkene coupling partners. To equally compare our newly developed conditions to the previously reported ones, we decided to run a control where 10 equiv of carbonyl was used in AcOH in the presence of 2.0 equiv of Mn(OAc) 3 at 70 °C. Strikingly, except for nitroacetate, our reaction conditions outperformed the control experiments. More importantly, for compounds 18, 28, 31, 34, and 37, our conditions always afforded products, while previously reported ones were ineffective. This observation motivated us to try to understand the reaction further to optimize for those low yielding substrates.

Rapid Optimization of Low Yielding Substrates.

We envisioned that we could re-apply the knowledge accumulated during our systematic study to identify key The Journal of Organic Chemistry 328 parameters that should impact the outcome of the reaction. If 329 successful, this approach would allow the practitioner to 330 quickly identify optimal reaction conditions to give at least 331 some product under the general stoichiometric set of 332 conditions for challenging substrates. 333 Throughout our initial investigation of the Mn-mediated α-334 radical addition of carbonyls to olefins, parameters such as 335 solvent, temperature, manganese loading, and concentration 336 were found to dramatically impact the reaction outcome 337 (Scheme 2). Due to its importance in previously reported 338 conditions by the Snider group, AcOH was also a variable that 339 needed to be considered. 11g With those parameters in mind, 340 eight sets of conditions were designed and assessed on eight 341 substrates containing seven ketones and two alkenes. Depend-342 ing on the substrates and the conditions used, huge reactivity 343 differences were observed matching with the observed 344 difficulty to develop a truly general protocol (vide supra). As 345 an example, cyanoester afforded high conversions to desired 346 product 23 under all tested conditions even at room 347 temperature. This extremely high reactivity can explain why 348 it is one of the most frequently used substrates in literature 349 reports. 24 Conversely, several changes impacted the conversion 350 to product 20 which results from the coupling between 351 nitroacetate and hexadecene. Running the reaction with more 352 equivalents of manganese (from 1.5 to 2.5 equiv), higher 353 temperature (from 70 to 90 °C), higher concentration (from 354 0.75 to 1 M), or using AcOH as an additive (5 or 10 equiv) 355 provided better conversions versus the original conditions. The 356 reactivity of α-tetralone, which already delivered reasonable 357 conversion (25%), was enhanced by simply adding 5 equiv of 358 AcOH to afford 56% of desired product 18. Similarly, 359 compound 38 was obtained in a moderate yield of 43% by 360 increasing the amount of manganese from 1.5 to 2.5 equiv.

361

While trying to optimize compounds 28, 34, and 39 proved 362 more difficult, several conditions still afforded decent 363 conversions (20-50%). Therefore, it was envisioned that the 364 combination of all promising set of conditions could 365 significantly increase the yield of initially low conversion 366 reactions. This merged approach was attempted on several s4 367 substrates, and the results are highlighted in Scheme 4B. 368 Outstandingly, compounds 34, 18, 28, and 20, which were 369 initially only observed in trace amounts or isolated in low 370 yields using the initial set of conditions, were obtained in 50, 371 49, 44, and 66% isolated yields, respectively. 372 Testing of these simple parameters (manganese loading, 373 temperature, concentration, and AcOH additive) can quickly 374 identify optimized conditions for challenging substrates. 375 However, we thought it would be even better if the practitioner 376 could predict the reaction outcome based on the nature of the 377 carbonyl and the olefin. 378 3.5. Predicting Reaction Outcome. The systematic 379 optimization, scope evaluation, and the subsequent re-380 optimization of low yielding compounds afforded copious 381 data on reaction outcomes and allowed for a predictive model 382 that relied on (1) the pKa of the carbonyl and (2) the 383 geometry and substitution of the olefin to be assembled 384 (Scheme 4C).

385

Several studies have highlighted the importance of steric 386 hindrance and pKa depending on the nature of the carbonyl 387 coupling partner. 11,25 However, no general trends have been 388 reported due to the complexity of the transformation and the 389 myriad of conditions associated to it. Based on our 390 stoichiometric set of conditions, the reaction outcome can be predicted by simply using the pKa of the carbonyl starting material (Scheme 4C.1). Carbonyl compounds with a pKa lower than 24 will provide good yields under the optimized stoichiometric conditions. For carbonyls with pKas between 24 and 29, the approach developed for the low yielding compounds should be applied to obtain satisfactory yields.

Compounds with pKas higher than 29 such as esters and amides will not deliver the desired product.

As highlighted by several reviews, the impact of the substitution and geometry of the alkene coupling partner has not been deeply studied. 11 Indeed, most of the examples reported in the literature focus on the functionalization of terminal nonsubstituted and cyclic olefins. The evaluation of the reaction scope and the optimization of its limitations allowed us to rationalize outcomes based on the nature of the olefin (Scheme 4C.2). Terminal alkenes and alkynes as well as stabilized disubstituted olefins are well tolerated under the reaction conditions and should afford good yields using the standard stoichiometric protocol. For nonstabilized, cyclic, and cis/trans alkenes, the eight conditions designed using the systematic study should deliver moderate yields. When the olefin is too encumbered, no desired product should be expected.

Development of an Electrocatalytic Set of

Conditions. With the stoichiometric set of conditions, the knowledge accumulated from the systematic study, and the ability to predict the reaction outcome, the practitioner will be able to confidently run the Mn-mediated α-radical addition of carbonyls to olefins. However, the need for a stoichiometric amount of Mn(OAc) 3 was still a serious limitation from a sustainability and scalability standpoint. The development of a catalytic version was therefore sought. As mentioned in the Introduction, electrochemical alternatives have been attempted

for Mn(III)-mediated reactions but have either not proven general, were not deeply investigated, or suffered from limitations such as the requirement for a divided cell and limited substrate scope. 15e,19,24b,26 Therefore, the development of a robust electrochemical version of the Mn-mediated carbonyl α-radical addition to olefins was undertaken.

The oxidation of Mn(II) to Mn(III) was first studied in an undivided cell equipped with a graphite anode and cathode s5 (Scheme 5A). The reaction was conducted using Mn(OAc) 2 as the catalyst in AcOH at room temperature under argon. AcOH was selected as the solvent due to the dual role it would ensure through the course of the catalytic reaction. While proton reduction would occur at the cathode, the subsequently generated acetate would assist the regeneration of the Mn(OAc) 3 active catalyst at the anode. After 1 F•mol -1 , the formation of Mn(OAc) 3 was clearly detected. However, passivation of the electrodes was also observed due to accumulation of the Mn-based species on the anode surface.

Pleasingly, this undesirable phenomenon was easily suppressed by simply applying alternating polarity. Indeed, such a mode allow for the polarity of each electrode to be switched at a defined frequency lowering charge accumulation.

With this proof of concept in hand, the carbonyl starting material was carefully selected. Indeed, cyanoacetate was an ideal coupling partner to start the optimization with as it allowed the reaction to be conducted at room temperature and tolerated the use of AcOH (vide supra, Scheme 4). The excess of carbonyl was maintained at 3 equiv, while the amount of Mn(OAc) 2 was fixed at 10 mol %. Based on our earlier optimization studies, the impact of the [Mn]/[AcOH] ratio on

The Journal of Organic Chemistry only delivered 49% of the desired compound, while using 10 mA instead of 5 mA also lowered the yield to 68%. To avoid scalability issues associated with the use of perchlorate-based electrolytes, LiBF 4 was used instead of LiClO 4 , which maintained high conversion to product 23 and good isolated yield (77%). Finally, aware of the beneficial effect of running the reaction at high concentration, the electrochemical conditions were concentrated up to 0.5 M (instead of 0.25 M), and compound 23 was isolated in 84% yield.

We then decided to synthesize several molecules to assess the efficiency of the electrochemical protocol (Scheme 5D).

Compound 40 bearing a halogen moiety was isolated in 40% yield. As expected, compounds 14 and 21 which usually require higher temperature to proceed in good yield did not give satisfactory results at room temperature. Simply heating these reactions to 70 °C afforded good yields (71% for 14 and 67% for 21). In addition, the reaction was easily scaled-up to 6 mmol in a single 20 mL ElectraSyn vial by adjusting the current to 25 mA. Finally, the knowledge gained throughout the study was easily transferable to the electrochemical protocol with compound 34 obtained in 59% by using 20 equiv of AcOH in toluene at 90 °C. Altogether, these results not only highlight the efficiency of the optimized paired electrochemical protocol but also demonstrate its scalability. [START_REF] Vantourout | From Bench to Plant: An Opportunity for Transition Metal Paired Electrocatalysis[END_REF]

SUMMARY

The key mechanistic features of the developed conditions are s6 summarized in Scheme 6. To increase the practicality of the Mn-mediated α-radical addition of carbonyls to olefins, the goal of this study was to lower the amount of the carbonyl starting material to 3 equiv. Fixing this parameter impacted the first step of the mechanism where the carbonyl moiety needs to coordinate the Mn-catalyst. Therefore, increasing the concentration (from 0.1 to 0.75 M) was crucial to obtain the desired product in good yield (Scheme 6, panel A). The following loss of proton is afforded through an intramolecular deprotonation process where the presence of the acetate ligands proved essential. Indeed, external bases or additives were detrimental for the reaction to occur with high efficiency (Scheme 6, panel B). The oxidation of the resulting enolate has been extensively studied in the literature and none of the reaction parameters we have investigated improved it (Scheme 6, panel C).

Conversely, for the free-radical addition step, our study systematically highlighted the importance of the alkene geometry and substitution (Scheme 6, panel D). The high The Journal of Organic Chemistry concentration also favored this step of the mechanism. The use of EtOH instead of AcOH, in addition to increasing the functional group tolerance, positively impacted the final HAT step (Scheme 6, panel E). Finally, the use of electrochemical conditions to reoxidize Mn(II) to Mn(III) allowed the development of a robust and general catalytic set of conditions where a precisely controlled [Mn]/[AcOH] ratio was found to be critical (Scheme 6, panel E).

CONCLUSIONS

An in-depth study of the Mn-mediated α-radical addition of carbonyls to olefins has been assembled, allowing the development of a new and general set of reaction conditions. A wide range of functional groups including ester, cyano, nitro, free, and TBS-protected alcohols and acetyl groups were tolerated, and several carbonyls and olefins proved competent coupling partners. Understanding the effect of solvent, concentration, temperature, amount of the catalyst, as well as the impact of additives has enabled us to identify a list of eight key experiments that can quickly be performed to optimize challenging reactions. This approach was successfully applied to four different initially low yielding substrates, proved highly efficient, and led to the creation of a predictive model based on the pKa of the carbonyl compound and both the substitution and geometry of the alkene coupling partner. Finally, development of a robust and practical electrocatalytic version of the reaction enabled the departure from stoichiometric manganese loadings. The e-procedure requires only 3 equiv of the carbonyl starting material to achieve good conversions and uses two inexpensive graphite electrodes and a safe electrolyte. Importantly, this paired electrochemical process does not require the use of an undivided cell and was straightforwardly scaled-up to 6 mmol in a single 20 mL vial. Overall, both

a

  Scheme 3. Scope of the Reaction: (A) Carbonyls, (B) Alkenes, (C) Limitations, and (D) Robustness Test a

Scheme 4 .a

 4 Scheme 4. Broadening the Scope of the Reaction: (A) Reengaging Data from the Systematic Study, (B) Rapid Optimization of Initially Low Yielding Compounds, and (C) Predicting Reaction Outcome a

  the reaction outcome was first investigated (Scheme 5B). As expected, a high [Mn]/[AcOH] ratio did not lead to sufficient concentration of the active catalyst and afforded the desired product in low yield. Conversely, too much AcOH in the reaction gave a low [Mn]/[AcOH] ratio and reduced the efficiency of the transformation. The optimal [Mn]/[AcOH] ratio was obtained using 7.5 equiv of AcOH which afforded the desired product 22 in 88% yield. With this optimized ratio in hand, we turned our attention to the electrochemical reaction conditions (Scheme 5C). To maximize the conversion, 15 mol % of the Mn(OAc) 2 catalyst was used in the presence of 11.3 equiv of AcOH to maintain the [Mn]/[AcOH] ratio (0.02). Under these conditions, the desired product was obtained in more than 95% yield. A control experiment without AcOH led to extremely low conversion. In the absence of alternating polarity, compound 23 was only obtained in 15% yield, supporting the detrimental impact of electrode passivation on the reaction outcome. Using 1.5 F•mol -1 instead of 3.0 F•mol -1

Scheme 5 .

 5 Scheme 5. Electrochemical Mn-Mediated α-Radical Addition of Carbonyls to Olefins: (A) e-Oxidation of Mn(II) to Mn(III), (B) Optimum [Mn]/[AcOH] Ratio, (C) Optimization, and (D) Scope a

  is available free of charge at 553 https://pubs.acs.org/doi/10.1021/acs.joc.2c00054.554Experimental procedures and graphical abstracts for 555 both the stoichiometric and catalytic reactions, inves-556 tigation of the reaction parameters, optimization of the 557 reaction conditions (stoichiometric and catalytic).vantourout@univ-lyon1.fr

https://doi.org/10.1021/acs.joc.2c00054 J. Org. Chem. XXXX, XXX, XXX-XXX

https://doi.org/10.1021/acs.joc.2c00054 J. Org. Chem. XXXX, XXX, XXX-XXX H

https://doi.org/10.1021/acs.joc.2c00054 J. Org. Chem. XXXX, XXX, XXX-XXX I