- Open Access
A de novo variant in ADGRL2 suggests a novel mechanism underlying the previously undescribed association of extreme microcephaly with severely reduced sulcation and rhombencephalosynapsis
- Myriam Vezain†1,
- Matthieu Lecuyer†1,
- Marina Rubio2,
- Valérie Dupé3,
- Leslie Ratié3,
- Véronique David3,
- Laurent Pasquier4,
- Sylvie Odent3, 4,
- Sophie Coutant1,
- Isabelle Tournier1,
- Laetitia Trestard5,
- Homa Adle-Biassette6, 7,
- Denis Vivien2,
- Thierry Frébourg1, 8,
- Bruno J Gonzalez1,
- Annie Laquerrière†1, 9 and
- Pascale Saugier-Veber†1, 8Email authorView ORCID ID profile
© The Author(s). 2018
- Received: 27 September 2018
- Accepted: 29 September 2018
- Published: 19 October 2018
Extreme microcephaly and rhombencephalosynapsis represent unusual pathological conditions, each of which occurs in isolation or in association with various other cerebral and or extracerebral anomalies. Unlike microcephaly for which several disease-causing genes have been identified with different modes of inheritance, the molecular bases of rhombencephalosynapsis remain unknown and rhombencephalosynapsis presents mainly as a sporadic condition consistent with de novo dominant variations. We report for the first time the association of extreme microcephaly with almost no sulcation and rhombencephalosynapsis in a fœtus for which comparative patient-parent exome sequencing strategy revealed a heterozygous de novo missense variant in the ADGRL2 gene. ADGRL2 encodes latrophilin 2, an adhesion G-protein-coupled receptor whose exogenous ligand is α-latrotoxin. Adgrl2 immunohistochemistry and in situ hybridization revealed expression in the telencephalon, mesencephalon and rhombencephalon of mouse and chicken embryos. In human brain embryos and fœtuses, Adgrl2 immunoreactivity was observed in the hemispheric and cerebellar germinal zones, the cortical plate, basal ganglia, pons and cerebellar cortex. Microfluorimetry experiments evaluating intracellular calcium release in response to α-latrotoxin binding showed significantly reduced cytosolic calcium release in the fœtus amniocytes vs amniocytes from age-matched control fœtuses and in HeLa cells transfected with mutant ADGRL2 cDNA vs wild-type construct. Embryonic lethality was also observed in constitutive Adgrl2−/− mice. In Adgrl2+/− mice, MRI studies revealed microcephaly and vermis hypoplasia. Cell adhesion and wound healing assays demonstrated that the variation increased cell adhesion properties and reduced cell motility. Furthermore, HeLa cells overexpressing mutant ADGRL2 displayed a highly developed cytoplasmic F-actin network related to cytoskeletal dynamic modulation. ADGRL2 is the first gene identified as being responsible for extreme microcephaly with rhombencephalosynapsis. Increased cell adhesion, reduced cell motility and cytoskeletal dynamic alterations induced by the variant therefore represent a new mechanism responsible for microcephaly.
- Human extreme microcephaly
At the end of the 4th post-conception week (PCW), the neural tube closes and immediately undergoes drastic changes, which consist in the setting of several events regulated by multiple, often redundant, signalling pathways leading to anteroposterior and dorsoventral polarity and emergence of four curvatures that demarcate the primary cerebral vesicles—the prosencephalon, the mesencephalon, pons and myelencephalon—from the spinal cord. Concomitantly, other key events come into play to allow the proper growth, folding and differentiation of all brain structures and particularly of the cerebral cortex; these events are schematically divided into three main stages encompassing cell proliferation with expansion of the progenitor population, neuronal migration and post-migration developmental processes. The critical role of these events, which are necessary for appropriate development and function of the human six-layered cortex, is reflected by the wide range of disease phenotypes arising from their disruption, the most severe of them being polymicrogyria, lissencephaly, microcephaly and microlissencephaly.
Lissencephalies are usually single-gene disorders that affect neuronal migration during cortical development; polymicrogyria, which has been associated with genetic and environmental causes, is still often considered as secondary to abnormal post-migration development [9, 21, 22, 33]. Microlissencephaly is a rare condition characterized by severe congenital microcephaly with absent sulci and gyri with either a thinned or thickened cortical plate. Similar to polymicrogyria, microcephaly and microlissencephaly may be due to both genetic and environmental causes, the latter including infections, toxic insults (notably antiepileptic drugs, opioids or cocaine) and prenatal alcohol exposure. Genetic causes are multiple and result from abnormal neuronal proliferation or survival associated with defective neuronal migration [5, 21]. Whatever the cause, lissencephaly and microlissencephaly may be observed alone or in combination with various brainstem or cerebellar lesions.
Among the diverse cerebellar developmental abnormalities, rhombencephalosynapsis (RES) is an extremely rare malformation initially described by Obersteiner as complete or partial vermis agenesis with fusion of the cerebellar hemispheres and apposition or fusion of the deep cerebellar nuclei . RES is thought to occur early during embryogenesis, between the 5th and 7th PCW, but its pathophysiological mechanism remains a matter of debate, considered by some authors as resulting from a fusion and by others from a non-separation of cerebellar hemispheres over an absent or severely hypoplastic vermis [8, 32, 56]. RES occurs in a vast majority of cases as a sporadic condition consistent with de novo dominant variations, and to date, exceedingly rare syndromic forms have been described and comprise Gomez-Lopez-Hernandez syndrome (MIM#601853), Fanconi anaemia complementation group B (MIM#300514) and autosomal recessive (MIM#276950) or X-linked (MIM#314390) inherited condition designated VACTERL-H . In sporadic forms, RES occurs in isolation or in combination with other central nervous system (CNS) and extra-CNS malformations; it has been described in association with mesencephalic lesions such as atresia forking of the aqueduct of Sylvius and fusion of the colliculi. Associated supratentorial lesions have also been reported, consisting in agenesis of the corpus callosum, atresia of the 3rd ventricle, holoprosencephaly and neural tube closure defects . So far, however, the association of severe microcephaly with RES has never been reported to our knowledge.
Using comparative patient-parents exome sequencing strategy, a powerful method to detect de novo pathogenic variants involved in human Mendelian genetic diseases [52, 53], we identified the first molecular basis of this association of extreme microcephaly with severely reduced sulcation with RES in a fœtus, a deleterious variant in the ADGRL2 gene, which encodes an adhesion G-Protein-Coupled Receptor (GPCR). Mechanistic and functional characterization of the variant provides compelling evidence that this deleterious variant causes early human developmental defects involving both supratentorial and infratentorial structures.
Whole exome sequencing
The parents provided written informed consent for Whole Exome Sequencing (WES). High quality genomic DNA was extracted from the peripheral blood of the fœtus and her parents using QIAamp DNA Blood Midi Kit (Qiagen, Courtabœuf, France) and QuickGene DNA Whole Blood Kit L (Kurabo, Japan), respectively, according to the manufacturer’s instructions. Approximately 3 μg was sheared with a Covaris E220 DNA Sonicator (Covaris, Inc., Woburn, MA, USA) and coding regions captured using a SureSelectXT Human All Exon V2 kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. The enriched libraries were sequenced on a Genome Analyzer IIx (GAIIx, Illumina, Inc., San Diego, CA, USA) with 76 bp paired-end reads. Image analysis and base calling were performed by Real-Time Analysis (RTA 1.10) and CASAVA software (v1.8, Illumina, Inc.). Reads were mapped to the human reference sequence (GRCh37, Hg19) with the Burrows-Wheeler Aligner (BWA v.0.6.2). Read duplicates were marked with Picard tools, local realignments around indels, base-quality-score recalibration and variant calling were performed with the Genome Analysis Toolkit (GATK 2.5). Single-nucleotide variants and small indels were identified with the GATK UnifiedGenotyper and were filtered according to the Broad Institute’s best-practice guidelines (Additional file 1: Table S1). Variants were then annotated with ANNOVAR (version 2012). Filtration of unknown variations and differential exome analysis were achieved using the Exome Variation Analyzer (EVA 2.0), our in-house software . To evaluate its pathogenic potential, the ADGRL2 DNA sequence alteration was analysed in the following web-based programs: MutationTaster , SIFT  and PROVEAN .
Sanger sequencing analysis
The 20 ADGRL2 exons, 100 bp exon-intron boundaries and UTRs were PCR amplified from 50 to 100 ng of genomic DNA extracted from peripheral blood (exome trio) and from fœtal tissues coming from the Department of Genetics, Rennes University Hospital. These DNA samples were first amplified using the Whole Genome Amplification GenomePlex2 kit (Sigma-Aldrich, St Louis, MO, USA). Sanger sequencing of these fragments was performed using the BigDye® Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, Courtabœuf, France). Sequencing reactions were migrated on a 3100xl Genetic Analyzer (Applied Biosystems) and analysed using the Sequencing analysis software 5.2.0 (Applied Biosystems). PCR and sequencing primers are available upon request.
Adgrl2 mouse and chicken in situ hybridization
Chick (Gallus gallus) or mouse (C57Bl6) embryos were fixed overnight at 4 °C in 4% paraformaldehyde (PFA), rinsed and processed for whole-mount RNA in situ hybridization. Chick embryos were staged according to Hamburger and Hamilton (HH) . For the hybridization step, embryos were permeabilized 5 min in proteinase K solution (10 μg/ml), then fixed for 20 min in 4% PFA/0,2% Glutaraldehyde. After several washes in PBT, the embryos were incubated in a prehybridization solution (50% formamide, 5× SSC pH 4.5, 2% SDS, 2% blocking reagent (Roche, Meylan, France), 250 μg/ml tRNA, 100 μg/ml Heparin) at 65 °C before the addition of 10 μg/ml of the Adgrl2 probe, and overnight incubation at 65 °C. Probes were generated by PCR, subcloned in pCRII-TOPO® (Invitrogen, Saint Aubin, France), and used to transcribe the digoxigenin (DIG)-labelled antisense RNA probes. After incubation, embryos were washed 4 times for 30 min with a solution containing 50% formamide, 2× SSC and 1% SDS at 65 °C, then cooled down to room temperature in 1 M maleic acid buffer containing Tween 20 (MABT) and washed several times. For the antibody step, nonspecific binding was blocked by incubating 2 times for 30 min then 1 h in MABT containing a 2% blocking reagent solution and 20% normal calf serum. AP-conjugated anti-DIG antibody was added at a concentration of 1:3000 and incubated overnight at 4 °C. The embryos were washed 5 times for 1 h in MABT at room temperature, followed by 2 times for 10 min in a solution containing 100 mM NaCl, 100 mM Tris-HCL, 50 mM MgCl2 and 0.1% Tween20 at pH 9.5 (NTMT). The AP-conjugated anti-DIG antibody was detected by a mixture of NBT/BCIP in NTMT, pH 9.5. The reaction was stopped by washing in PBT once the required staining intensity was achieved.
ADGRL2 immunohistochemical studies in normal human embryos and fœtuses
A series of 3 embryos and 19 fœtuses were selected for this study (collection number DC-2015-2468, cession number AC-2015-2467). Detailed characteristics of the selected cases are presented in Additional file 2: Table S2. Gestational age was estimated according to biometric data, skeletal measurements and histological maturation of the brain and viscera. Six-μm paraffin-embedded sections from whole embryos (6–10 PCW) and from brains and gonads from fœtuses at 13 weeks of gestation (WG) to birth were mounted on coated slides (Superfrost Slides, Thermo Fisher Scientific, Illkirch, France) and dried overnight in a convection oven (37 °C). Induced epitope retrieval was performed by immersion in a citrate buffer solution pH 6 at 95 °C for 1 h. Incubations with the primary antibody ADGRL2 (diluted 1:200, Clinisciences, Nanterre, France) were carried out for 1 h at room temperature using the Benschmark Ultra system (Ventana Medical Systems, Tucson, AZ), the primary antibody being diluted in an antibody diluent reagent solution (Life technologies, Saint Aubin, France). After incubation, slides were processed by the detection kit Ultraview (Ventana Medical Systems). Peroxidase was visualized using the alkaline phosphatase detection kit (Ventana Medical Systems). Slides were rinsed in tap water, counterstained with hæmatoxylin and mounted in mounting medium. Negative controls were obtained by omission of the primary antibody or by the use of other antibodies of known reactivity.
Amniocytes from control and patient fœtuses were collected by amniocentesis at 19 WG in order to explore chromosomal abnormalities. HeLa and amniocytes cultures were grown as monolayer in T-75 flasks. Cells were incubated in Ham’s F12 nutrient mixture (Gibco, Life Technologies, Saint-Aubin, France) containing 10% fœtal bovine serum (Gibco) and 2 mM L-Glutamin (Sigma-Aldrich) at 37 °C in an atmosphere of 5% CO2.
Amniocytes were washed once with 1× phosphate saline buffer (PBS), trypsinized, centrifuged at 1,500 × rpm for 5 min, and solubilized in 100 μl of RIPA buffer (Thermo Fisher Scientific) with 1× Protease Inhibitor Cocktail (Sigma-Aldrich) and 1× Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) for 30 min at 4 °C. After 30 min of centrifugation at 14,000×g, 30 μg proteins were separated by denaturing sodium-dodecyl sulphate polyacrylamide gel electrophoresis (10%, SDS-PAGE) and transferred to nitrocellulose membrane (Hybond C-Extra; Amersham Biosciences, Arlington Heights, IL, USA). Blots were blocked for 1 h with 5% skimmed milk in PBS and incubated with anti-ADGRL2 polyclonal antibody (1:500, LifeSpan BioSciences, Seattle, WA, USA) or anti-GAPDH polyclonal antibody (1:1000, Abcam, Cambridge, UK) in 0.05% Tween-PBS (PBST) overnight at 4 °C under gentle agitation. Membranes were washed with PBST, and primary antibody was detected using peroxidase-labelled anti-rabbit or anti-goat antibodies (1:10,000, Jackson Immunoresearch Laboratories, West Grove, PA, USA). Signals were detected with chemiluminescence reagents (Pierce Biotechnology, Rockford, IL, USA) and acquired with a G:BOX (Syngene, Cambridge, UK), monitored by the Gene Snap software (Syngene). The signal intensity in each lane was quantified using the Genetools software (Syngene), and the ratio of ADGRL2 signal vs GAPDH was calculated. All immunoblotting experiments were carried out in triplicate.
Plasmids and cell transfection
The Adgrl2 variation was introduced using Quick Change XL Site-Directed Mutagenesis Kit (Agilent Technologies), into previously published pcDCIRL-2 or pcDCIRL-2-GFP expression plasmids containing the full length rat Adgrl2 cDNA . Owing to the fact that rat wild-type Adgrl2 protein contains a phenylalanine (TTC) instead of the leucine (CTC) at position 1262 in humans (same hydrophobic class, Grantham distance 22), we performed a double mutagenesis to introduce the mutant-related histidine (CAC) at position 1262. Wild-type and mutant pcDCIRL-2 expression plasmids (2 μg) were transiently transfected into HeLa cells, using fuGENE 6 transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Cells were plated at 5 × 105 cells/well in 6-well plates in Ham’s F12 medium, and transfections were incubated for 48–72 h to allow Adgrl2 addressing to the plasma membrane. All transfection experiments were carried out in triplicate.
Microfluorimetry and intracellular calcium measurements
For measurement of whole cell intracellular calcium levels, amniocytes or HeLa cells were grown in 6 well plates on glass coverslips (diameter 30 mm) coated with poly-L-lysine (Sigma-Aldrich). Cultured cells were rinsed in PBS and incubated in Ham’s F12 culture medium containing 10 μM of the calcium probe Fura-2 AM, 0.3% pluronic F-127 (Molecular Probes, Life Technologies, Cergy-Pontoise, France) and 50 μM MK-571 sodium salt hydrate (Sigma Aldrich) for 30 min at 37 °C in the dark. After the loading step, cells were washed twice for 5 min in culture medium, and the coverslips supporting the cells were placed under an inverted fluorescence Leica DM 6000B microscope equipped with a rapid shutter wheel (Rueil-Malmaison, France) and continuously infused with Ham’s F12-EDTA solution. The fluorescent signals associated with calcium-free and calcium-bound Fura-2 were acquired by alternately exciting the cells at 340 and 380 nm. The emitted fluorescence was collected at 510 nm and a ratio of both signals was calculated using the Metamorph software (Roper Scientific, Evry, France). Total recording time was 15 min, and the time interval between two acquisitions was 5 s. After 3 min baseline recording, α-latrotoxin (1 nM, Alomone Labs, Jerusalem, Israel) was added in the calcium-free perfusion medium. To allow dimer formation, α-latrotoxin was prepared in Ham’s F12 calcium free medium 30 min before use. After a 3-min period under drug stimulation, the perfusion medium was replaced by Ham’s F12 containing 1.2 mM calcium. The exogenous ligand α-latrotoxin induces intracellular Ca2+ (Ca2+i) elevation by two mechanisms that are not mutually exclusive, i.e., activation of phospholipase C (PLC) through specific ADGRL receptor activation [4, 42] and the ionophoric properties of α-latrotoxin [3, 30]. Culture media complemented with 4 nM EDTA were used to induce extracellular Ca2+ chelation and target intracellular calcium flux resulting from ADGRL receptor activation . When required, cells were incubated during the loading step with 10 μM of the phospholipase C inhibitor U73122 (Sigma Aldrich) . Data were then exported to the biostatistics Prism software (GraphPad Inc., San Diego, CA) and areas under the curve were quantified. For statistical analyses, experiments were performed three times on at least 20 cells per condition.
Cell cycle analyses in human fœtal ganglionic eminences
Since the germinal zone of the dorsal telencephalon disappears by 24WG, proliferative stem cells were evaluated on lateral ganglionic eminences (LGE), which are known to massively produce interneurons at this stage . LGE were micro-dissected from the paraffin-embedded section passing through the diencephalon from the fœtus, from an age-matched control brain aged 26WG as well as from a fœtus interrupted for microcephaly due to homozygous pathogenic variant in the MCPH1 gene, NM_024596.3:c.427dup;p.(Thr143Asnfs*5). MCPH1 variations are responsible for delayed mitosis of cycling progenitors in the germinal zones, and in microlissencephaly brain lesions may also include extreme hypoplasia of the cerebellum and brainstem. The sections were treated using the technique described by Hedley et al. with minor modifications . Briefly, 35-μm sections were dewaxed in xylene and rehydrated in decreasing concentrations of ethanol. Enzymatic digestion was carried out in a 0.5% pepsin PBS solution. The nuclear suspension, adjusted at a final concentration of 106 nuclei/ml were incubated in a 1 mg/ml ribonuclease solution for 30 min at 37 °C and stained with propidium iodide (6 μg/ml in a 40 μM trisodium citrate solution) for 1 h at 4 °C. Flow cytometric analyses of the cell cycle were performed on an XL Beckman Coulter flow cytometer (Coultronics, Hialeah, Florida, USA) with the 488-nm wavelength of an ion argon laser as an excitation source. The cytofluorograph was adjusted to maximal resolution with flow check microspheres (Coultronics) having a coefficient of variation lower than 1.5%. For each sample, 104 to 105 cells were analysed, and the proliferative index (PI) was estimated using the Multicycle software (Coultronics), calculated as the percentage of cells in phases S + G2/M.
Cytometric analyses of Adgrl2 transfected HeLa cells
HeLa cells were transfected with an empty vector or with plasmids encoding the wild-type or the mutant Adgrl2 cDNA. After a 3-day culture, cells were gently detached from their support using 1 mM EDTA in PBS and processed for cell cycle analysis according to the three steps method of Vindeløv . Single cell suspensions were obtained after incubation in a 3% trypsin PBS buffer solution. Cell suspensions were filtered through a 48-μm pore nylon gauze. Cell count was adjusted to obtain a final concentration of at least 104 cells. The suspensions were mixed with chicken red blood cells (CRBC) used as internal standard, as described by Vindeløv et al. . The concentration was adjusted to obtain a final ratio of CRBC to cultured cells of 1/10. An aliquot of 300 μL of these nuclear suspension samples was centrifuged at 500 g, then resuspended in 500 μl of a staining solution including RNase, propidium iodide and non-ionic detergent Nonidet P40 for 10 min at 4 °C. Evaluation of the cell cycle was performed as described above. For evaluation of cell size and content, 5000 to 6000 cells were incubated in a PBS solution containing 0.3% saponin, 1% bovine serum albumin, 1% RNase and 0.005% propidium iodide. The size corresponding to the light emitted by the cells under exposition to the incident light of the laser (forward scatter) was arbitrarily expressed by the ratio number of cells vs fluorescence intensity on a scale of 1024 channels (AU, arbitrary units). The cellular content corresponding to the more or less heterogeneous cytoplasmic content (granulometry) was evaluated using emitted scattered light of the cell at 90° (side scatter) also expressed by the ratio number of cells vs fluorescence intensity on a scale of 1024 channels.
To study the addressing of Adgrl2 to the plasma membrane, HeLa cells were grown in 6-well plates on glass coverslips (diameter 10 mm) coated with poly-L-lysin (Sigma-Aldrich). HeLa cells were transfected with 2 μg pcD-empty vectors or with pcD plasmids encoding Wt and mutant rat Adgrl2 (CIRL-2) constructs coupled with a GFP tag using the fuGENE 6 transfection reagent (Promega). After 72 h, glass coverslips were rinsed with PBS and cells were fixed with 4% paraformaldehyde in PBS for 15 min. After 3 gentle washes with PBS, coverslips were mounted in DAPI-containing Vectashield (Vector laboratories, Cambridgeshire, UK) and images were acquired with the Leica laser scanning confocal microscope TCS SP2 AOBS (Leica Microsystems, Wetzlar, Germany).
Cell adhesion assays were performed in triplicate with HeLa cells as previously described . HeLa cells were transfected with 2 μg pcDCIRL-2 Wt or pcDCIRL-2 Mt. coupled or not with a GFP tag using fuGENE 6 transfection reagent (Promega). After 72 h, the living and dead cells were respectively labelled with cell tracker green (Invitrogen) and 7-amino-actinomycin D (7-AAD, Sigma Aldrich). Cells were gently detached using 1 mM EDTA in PBS and 5.105 cells were resuspended in 330 μL of aggregation medium (Ham’s F12 containing 10% FBS, 50 mM Hepes-NaOH, pH 7.4, 10 mM CaCl2, and 10 mM MgCl2) using a Countess Automated Cell Counter (Invitrogen). Cell suspensions were placed into 0.5-ml polypropylene tubes, leaving a small air bubble between the liquid and the lid and incubated under gentle agitation at 25 °C. Cell aggregation was addressed at T0 and T90 min by removing aliquots, spotting them onto culture slides (BD Falcon), and imaging the aggregates with a Leica DM 6000B microscope. Acquired images were then analysed by quantifying the number and size of aggregates using the Metamorph software (Roper Scientific). The mean aggregation index was calculated using the formula: (total aggregate area / aggregate number)T90 − (total aggregate area / aggregate number)T0. When required, the aggregation index of the HeLa cells was determined in the presence of 1 nM α-latrotoxin or 3 μM U73122 added in the aggregation medium.
Wound healing assay
HeLa cells transfected with 2 μg pcD-empty, pcDCIRL-2 Wt or pcDCIRL-2 Mt. plasmids were seeded in 6-well plates at a density of 5 × 105 cells per well. When cells reached confluence, a scratch was performed with a sterile tip to create an artificial wound. Tiff format images were acquired every 6 h during 72 h using an inverted microscope (Leica DM 6000B). Cell migration from the wound edge into the wound space was recorded and a time-course quantification of the scratch width was evaluated using the Metamorph software (Roper Scientific). Cell wound repair was calculated using wound width (expressed in percentage of the initial size).
Cytoskeletal network immunolabelling
HeLa cells were grown in 6-well plates on glass coverslips (10 mm diameter) coated with poly-L-lysin (Sigma-Aldrich). Cells were transfected with 2 μg pcD-empty or pcD plasmids encoding Wt or Mt. rat Adgrl2 (CIRL-2) using fuGENE 6 transfection reagent (Promega). After 72 h, glass coverslips were rinsed with PBS and fixed 15 min with 4% paraformaldehyde in PBS. HeLa cells were incubated overnight at 4 °C with an anti-acetylated α-tubulin monoclonal antibody (T-9026; Sigma-Aldrich) and Texas Red®-X Phalloidin (Molecular Probes) in the incubation buffer (PBS containing 1% bovine serum albumin (BSA) and 3% Triton X-100). Cells were rinsed twice with PBS for 20 min and incubated with the same incubation buffer containing the appropriate secondary antibody. Nuclei were visualized by incubating the cells for 5 min with a PBS solution containing 1 μg/mL Hœchst 33258. Fluorescent signals were observed with a Leica DMI 6000B microscope. The specificity of α-tubulin immunolabelling was controlled by omitting the primary antibody.
Mouse Adgrl2 inactivation
Adgrl2+/− mice were purchased from INFRAFRONTIER (Neuherberg, Germany). Adgrl2 was inactivated by insertion of a LacZ-neo cassette in the DNA sequence (strain B6;129P2-Adgrl2tm1Dgen/H, Mary Lyon Centre, MRC Harwell, Oxfordshire, UK). Mice were maintained at the animal facility of the Rouen Medical University. All rodent work was performed with the consent of the Animal Ethics Committee, Rouen Faculty of Medicine. All animals were fed a standard diet and maintained in a pathogen-free environment on a 12 h light/12 h dark cycle.
For genotyping, at least 3 mm of the mouse tail was cut, genomic DNA was then extracted using the NucleoSpin® Tissue kit (Macherey-Nagel, Hœrdt, France) and quantified using a NanoVue TM spectrophotometer (GE Healthcare Life Sciences, Pittsburgh, PA, USA). To make sure that mice were accurately genotyped, three genotyping assays were developed, the first multiplex PCR being designed to simultaneously detect the wild-type and targeted allele, the second being designed to detect the targeted allele only and the third to amplify the wild-type allele only. PCR primers and conditions are available upon request.
Magnetic resonance imaging (MRI)
To evaluate long-term brain lesions, MRI analyses were carried out on female and male adult (P45) mice using a Pharmascan 7 T (Bruker, Wissenbourg, France). T2-turboRARE 3D weighted images were acquired using a multislice multiecho sequence: TE/TR 31.5 ms/1500 ms. Quantification of morphometric brain characteristics was done using ImageJ (NIH software v1.45r, National Institute of Health, Bethesda, MD, USA) previously upgraded with the Bruker plugins. Image analyses provided access to qualitative and quantitative neuroanatomical criteria including cerebrum width, cerebrum volume, lateral ventricle volume and mid-sagittal anteroposterior vermis diameter.
Statistical analyses were performed using the biostatistics Prism software (GraphPad Inc.). Tests used for each experiment, the number of independent experiments and p-values are detailed in Additional file 3: Table S3.
Neuropathological examination reveals extreme microcephaly and RES
Whole exome sequencing reveals a de novo variation in the ADGRL2 gene
ADGRL2 resequencing in a panel of 29 unrelated RES-affected individuals fails to detect any pathogenic variant
As RES was one of the main lesions observed in the fœtus, the hypothesis that ADGRL2 variants could be responsible for a wider phenotypic spectrum was raised. All coding exons of the ADGRL2 gene by means of the Sanger technique were sequenced in 29 unrelated fœtuses affected with RES alone or with associated mesencephalosynapsis (atresia-forking of the aqueduct of Sylvius and fusion of the colliculi), diencephalosynapsis (atresia of the 3rd ventricle with collapse of the thalami), holoprosencephaly or encephalocele . No variant was detected in these 29 fœtuses.
Adgrl2 is early expressed during chicken and mouse development
Similar Adgrl2 expression domains were observed in mouse embryos at E9.5. Mouse Adgrl2 was strongly expressed in the telencephalon, mesencephalon and cerebellum, but absent in the diencephalon and at the mesencephalon-metencephalon boundary (Fig. 4c). These first expression studies of Adgrl2 reveal that mouse and chicken Adgrl2 display similar region-specific expression in the cephalic vesicles and that Adgrl2 is involved in a highly conserved mechanism that is crucial during early development of the telencephalon and cerebellum.
ADGRL2 is expressed from early human development
In human embryos, at 6th, 9th and 10th PCW, strong immunoreactivity was observed in almost all organs and tissues, notably in the liver parenchyma, heart, primary bronchi, digestive epithelium, nephrogenic blastema, smooth and striated muscle cells, vascular endothelium, as well as in mesenchymal tissues, especially the cartilaginous cells of the head, neck, thorax and of the axial skeleton. ADGRL2 immunoreactivity was strong in the seminiferous cords of the testes and in the epithelium of the epididymis from the 6th PCW, and from the 10th PCW in ovary germ cells. From 14WG onward, oogonia and follicular cells were intensively immunoreactive along with the ovarian superficial epithelium. From 18WG to birth, diffuse immunolabelling persisted in the primordial follicles (oogonia and follicular cells, Additional file 4: Figure S1a, b), and in the Leydig cells of the ovarian hilum (Additional file 4: Figure S1c). In male fœtuses, spermatogonia, Sertoli and Leydig cells, as well as interstitial mesenchymal testicular matrix, were strongly immunolabelled from 18WG to birth (Additional file 4: Figure S1d).
ADGRL2 expression is normal in patient amniocytes carrying the c.3785T>A variant
ADGRL2 is post-transcriptionally processed, the full length precursor being autoproteolytically cleaved in two independent fragments (N-terminal fragment, 70-75 kDa and C-terminal fragment, 105-110 kDa) which are addressed to the plasma membrane . To investigate whether the missense variant identified by WES could alter ADGRL2 protein stability or processing, protein expression was evaluated by western blot experiments on the patient amniocytes collected at the time of amniocentesis performed at 21WG, by comparison with amniocyte ADGRL2 expression from control fœtuses at the same developmental stage. As shown in Additional file 5: Figure S2, no differences in ADGRL2 expression and cleavage were observed between the patient’s and the control amniocytes, indicating that the ADGRL2 c.3785T>A variant does not alter ADGRL2 expression or processing, but rather impairs its functionality.
Intracellular Ca2+ release in response to ligand activation is altered in patient ADGRL2 amniocytes
To investigate in greater details the Ca2+i mobilization associated with the ADGRL2 receptor, Wt and Mt. cells were co-incubated in the presence of the PLC inhibitor, U73122 (Fig. 6b, c) . In F12-EDTA condition, 10 μM U73122 abrogated the cytosolic Ca2+i increase induced by α-latrotoxin in both Wt and Mt. cells (Fig. 6b, c). In contrast, U73122 had no significant impact on the late phase of Ca2+i increase resulting from EDTA removal (Fig. 6d).
ADGRL2 c.3785T>A variant is responsible for signal transduction alteration
To demonstrate the deleterious effect of the variant independently from the patient genetic background, HeLa cells were used as an exogenous cellular model in which the candidate gene was overexpressed, due to the very low expression of ADGRL2 in these cells. As human ADGRL2 cDNA is toxic to bacteria, cloning was not possible and a previously described construct derived from rat Adgrl2 homolog, pcDCIRL-2 coupled to GFP, was used . To generate mutant Adgrl2 clones, a histidine was introduced at position 1262 using site-directed mutagenesis. First, the correct processing and trafficking of the GFP coupled receptor to the plasma membrane of transfected HeLa cells was verified by confocal microscopy and transfection efficiency by western blot (Additional file 6: Figure S3a, b). However, in F-12 EDTA condition, microfluorimetry experiments performed with pcDCIRL-2-GFP cells did not display any cytosolic Ca2+i increase after α-latrotoxin exposure: only a late phase of Ca2+i increase was observed. Because C-terminal GFP could induce steric hindrance and inhibit Adgrl2 signal transduction coupled to G protein in response to ligand binding, the wild-type pcDCIRL-2 construct lacking the GFP sequence was transfected into HeLa cells. The resulting cytosolic Ca2+i profiles after α-latrotoxin exposure were very similar to those obtained with Wt amniocytes (Fig. 6a,). In contrast, HeLa cells overexpressing mutant Adgrl2 presented exclusively the late-phase Ca2+i increase (Additional file 6: Figure S3c), confirming that ADGRL2 c.3785T>A variant is responsible for the alteration of the signal transduction coupled to G-protein in response to ligand binding.
Adgrl2 −/− mice die at E15.5
An Adgrl2 knock-out (KO) mouse model was used to outline the phenotype associated to ADGRL2 inactivation. Adult heterozygous mice were fertile and survived for more than 1 year, but had a shorter fertility lifespan compared with wild-type mice, a few months vs 2 years, respectively. Adgrl2+/− F2 mice were mated in order to obtain Adgrl2−/−. A balanced sex ratio distribution was observed in the 15 litters analysed. Nevertheless, Adgrl2 genotyping of mouse pups was not in agreement with the Hardy–Weinberg equilibrium, confirming embryo lethality of Adgrl2−/− mice as previously described by the provider, who suggested an embryonic lethality at ~E15.5 (χ2 = 49.35). In contrast, E15 embryos Adgrl2 genotyping was concordant with the Hardy–Weinberg equilibrium (χ2 = 1.81), even if a slight deviation began to occur.
MRI findings in Adgrl2 +/− adult mice confirm microcephaly, hypoplasia of the vermis and mesencephalon
ADGRL2 c.3785T>A variant increases cell adhesion properties and reduces cell motility
Mutant ADGRL2 cells present size and cytoskeletal network alterations
One of the mechanisms required for cell adhesion consists in fluctuations of Ca2+i, which regulate the dynamic assembly of cytoskeletal elements [45, 62]. To visualize F-actin and microtubule network modifications, glass coverslips cultured HeLa cells overexpressing pcD-empty, pcDCIRL-2 Wt or pcDCIRL-2 Mt. were immunolabelled with phalloidin and acetylated α-tubulin (Additional file 8: Figure S5). Depending on the transfected plasmid, cells harboured various morphologies: pcD-empty cells possessed a fusiform shape (Additional file 8: Figure S5a) while pcDCIRL-2 Wt spread out on the substratum (Additional file 8: Figure S5b). In the pcDCIRL-2 Mt. condition, cells were significantly more spread out than pcDCIRL-2 Wt cells (Additional file 8: Figure S5c). However, when detached from their culture support, cells from these three conditions displayed only minor differences concerning their size and content. Using flow cytometry quantitative studies, HeLa cultured cells had a mean size of 234.2 ± 5.95 AU, HeLa cells transfected with an empty vector a mean size of 241.7+/− 8.30 AU, HeLa cells transfected with the Wt Adgrl2 a mean size of 238.7 ± 6.73 AU and cells transfected with the ADGRL2 variant a mean value of 258.6 ± 13.49 AU. Regarding intracellular heterogeneity, some differences were observed between HeLa cells alone (236.7 ± 3.65 AU) and cells transfected with the ADGRL2 variant (264.9 ± 5.70 AU). From these data, increased size of cells overexpressing Adgrl2 (Additional file 8: Figure S5b, c) appears to be directly associated with their over-adhesive properties. Similar to flow cytometry studies, which evidenced intracellular heterogeneity differences among the different conditions, immunohistochemistry revealed cytoskeleton architecture modifications (Additional file 8: Figure S5a-c). In the three conditions, the α-tubulin network was located in the perinuclear area, whereas the F-actin network appeared to be modified in the case of Adgrl2 overexpression. In control cells, a well-defined F-actin filament network was essentially located at the periphery of the cell (Additional file 8: Figure S5a). When Adgrl2 was overexpressed, cells displayed a highly developed cytoplasmic F-actin network with numerous anchoring points to the glass coverslip (Additional file 8: Figure S5b; arrows). Overexpression of the mutant Adgrl2 exacerbated this phenomenon in the cell surface membrane of HeLa cells by homophilic Adgrl2 interaction activation (Additional file 8: Figure S5c). Thus, the ADGRL2 c.3785T>A;p.(Leu1262His) variant that alters signal transduction coupled to G protein is responsible for excessive adhesion properties associated with abnormal cytoskeletal remodelling of cells overexpressing this variant.
Within the large GPCR superfamily, ADGRL2 (previously named LPHN2) together with ADGRL1 (previously named LPHN1), ADGRL3 (previously named LPHN3) and ADGRL4 (previously named ELTD1) belong to the Adhesion family encompassing 33 mammalian members . Adhesion-GPCRs are involved in several key molecular and cellular functions, including planar cell polarity, regulation of cytoskeleton organization, cell adhesion and migration, cell cycle, cell death and differentiation , but the precise mechanisms by which ADGRL2 acts remain elusive. An exogenous agonist for ADGRLs has long been identified: α-latrotoxin, the major neurotoxin in black widow spider venom, which attests a synaptic role for ADGRLs . Endogenous ligands include Teneurin-2 (also known as Lasso), neurexins and FLRT1–3 (Fibronectin Leucine-Rich Transmembrane protein) [11, 48, 54, 63]. Contrary to ADGRL1 and ADGRL3, ADGRL2 does not bind to neurexins (neurexin-1a, −1b, −2b), binds only weakly to teneurin-2, and interacts with FLRT3, but not with FLRT1. Because ADGRLs were first identified as putative synaptic receptors for α-latrotoxin, researchers initially focused their attention on the role of ADGRL2 in the synapse, showing that ADGRL2 could mediate synapse recognition and assembly and/or contribute to a synapse maintenance signal .
The c.3785T>A;p.(Leu1262His) variant is localized in the intracellular domain of ADGRL2, which exhibits 35% and 49% similarity between ADGRL1 and ADGRL3 proteins, respectively. G protein–mediated intracellular signalling has been demonstrated for ADGRL1 by its interaction with Gαo and its binding to teneurin-2, which induces Ca2+ signals [42, 63]. Microfluorimetry experiments confirmed that the c.3785T>A variant impairs the early stage of cytosolic Ca2+i release in response to α-latrotoxin binding, both in the patient’s amniocytes and in HeLa cells transfected with the mutant Adgrl2 construct. Moreover, addition of the PLC inhibitor U73122 in wild-type amniocytes induced early step calcium release impairment. Thus ADGRL2 activation is necessary for G protein–mediated intracellular signalling. More precisely, ADGRL2 is required for this early step of calcium release, whereas α-latrotoxin tetramer pores are responsible for passive Ca2+e influx into the cell during the second step. Pure α-latrotoxin is a very stable homodimer, which further assembles into tetramers in the presence of Mg2+ or Ca2+ to form α-latrotoxin pores . Cyclic nucleotide signalling and Ca2+ are known to be intracellular downstream targets for many extracellular guidance molecules. They convert the information from locally expressed guidance molecules to intracellular effectors, which control migration by regulating cytoskeleton dynamics, in particular the F-actin network. Using cerebellar granule cell cultures, Komuro et al. demonstrated that their migration speed from the transient external granule cell layer was correlated to both the amplitude and frequency of Ca2+ elevations, and that the reduction of the Ca2+ transients resulted in the termination of granule cell migration . The modulation of intracellular Ca2+ release by the adhesion-GPCR ADGRL2 could therefore exert a role in the regulation of neuronal migration. Cell adhesion assay confirmed that the inhibition of the G protein–mediated intracellular signalling was correlated with enhanced adhesion and increase in size of aggregates overexpressing mutant Adgrl2. Transfection of either wild-type or mutant-type pcDCIRL-2, the rat homologue of ADGRL2, in HeLa cells induced a strongly developed microtubule network with many focal adhesion points, indicating that ADGRL2 inactivation leads to increased adhesion properties due to intracellular Ca2+ flux and cytoskeletal organization perturbations. Further characterisation of focal adhesions points could provide additional evidence of cell migration alteration as the recruitment of talin and vinculin is correlated with the mechanical force applied to the focal adhesion .
The causal effect of the c.3785T>A;p.(Leu1262His) variation in ADGRL2 on the fœtal brain malformation in Adgrl2+/− mice, Adgrl2−/− genotype being lethal in utero at E15.5 was supported by MRI findings. Both male and female Adgrl2+/− adult mice displayed microcephaly, affecting mainly the telencephalon, although more severe in Adgrl2+/− female mice, with a defect in anteroposterior growth of the vermis, in line with ADGRL2 expression during telencephalic and cerebellar development in human embryos and fœtuses as well as in chicks and mice.
Our results suggest that this ADGRL2 variation impedes the proper development of the cerebellum resulting in RES which is thought to occur when the cerebellar primordium develops and probably results from abnormal function of genes expressed during initial patterning of the mesencephalon-rhombencephalon [43, 68]. For some authors, RES is thought to result from loss of anterior cerebellar anlage cells derived from the medial ventricular zone (VZ) of the cerebellar primordium destined to become the vermis, or from a shift of these anterior cells toward a more posterior and/or ventral hemispheric fate . For others, RES results rather from a loss of posterior expansion of the medial region of the cerebellar primordium . Expansion of the medial cerebellar primordium plays a considerable role in generating the vermis and the hemispheres, which grow together rapidly from the two other germinal zones, the rhombic lips. Although the precise mechanisms that lead to the proper individualisation of the vermis and the hemispheres (which are lacking in RES) remain unknown, ADGRL2 variation likely avoids expansion of the vermis and of the hemispheres from the medial VZ and EGL. Besides, the ADGRL2 ligand, FLRT3, has a strong specific expression in the isthmic organizer, whereas ADGRL2 is not expressed in this area . This non-overlapping pattern of expression could be due to a specific dosage code to specify the boundaries of the future cerebellum along the rostrocaudal axis of the rhombencephalon. Interestingly, it has also been shown that lat-1, an orthologue of vertebrate ADGRLs in C. elegans, plays an essential role in anterior-posterior tissue polarity in the embryo [41, 51]. Moreover, in the absence of lat-1, stem cells display positioning defects as they remain clustered near their place of birth, highly reminiscent of what observed in ADGRL2 mutant cells using wound healing experiments . Our results suggest that RES could result from abnormal positional cues along the anteroposterior axis.
Primary congenital microcephaly and microlissencephaly have classically been classified as disorders of neurogenesis, consisting above all in a severe decrease of symmetric and asymmetric divisions in the ventricular and subventricular zones resulting in neuronal cell production depletion. These disorders could be attributed to four major pathophysiological mechanisms, including first, early events (during the 5th gestational week) that have been postulated as being of major importance for the control of cell cycle and proper functioning of the primary cilium; second, abnormalities during the different phases of mitosis (prometaphase, metaphase and cytodieresis); third, anomalies of formation and positioning of the mitotic spindle; and fourth, abnormal structure and function of associated molecules that are involved in cytoskeletal dynamics.
A constellation of disease-causing genes have been described as affecting the early steps of neural tube development, i.e., planar polarity, primary cilium structure (such as kinesins) or functions involving in particular Wnt and Sonic hedgehog pathways, as well as cell cycle length controlling molecules, notably PAX6 and FLNA .
As regards the different phases of mitosis, WDR81 deleterious variants were shown to increase the number of mitotic cells with an accumulation of cells in prometaphase and metaphase resulting in mitotic delay without any impact on mitotic spindle or primary cilia organization . Variations in the CIT gene encoding citron kinase localizes to the cleavage furrow and midbody, where it functions in cytodieresis. The neuropathological phenotype includes extreme microcephaly or microlissencephaly .
In fact, the vast majority of genes responsible for microcephaly encode centrosomal proteins involved in spindle orientation or proteins regulating these centrosomal proteins. To date, bi-allelic deleterious variations in 13 genes (MCPH1, WDR62, CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135, CEP152, ZNF335, PHC1, CDK6, CENPE [20, 57]) which are normally expressed in ventricular (apical) and subventricular (basal) radial glial cells, impair proper spindle positioning and orientation leading to a drastic decrease in the generation of intermediate progenitors and of post-mitotic neuroblasts due to a lack of maintenance of centrosome asymmetry . In these pathological conditions, brain lesions range from microcephaly with simplified gyral pattern to microlissencephaly. Pathogenic variations in NDE1 which interacts directly with LIS1 and is expressed in the neuroepithelium of the cerebral and cerebellar cortices where it contributes to interkinetic nuclear migration and also localized at the centrosome and on the mitotic spindle, result in early failure of neuron production and microlissencephaly [1, 7].
Microtubule structure alterations as well as alterations of binding partners necessary for proper microtubule dynamics resulting from pathogenic variants in TUBA1A, TUBB2A, TUBB2B, TUBB3, TUBB5, TUBG1 and TUBA8 genes are also responsible microcephaly and microlissencephaly. These lesions are constantly associated with several brain abnormalities due to defects in progenitor proliferation with decreased symmetric divisions, impaired neuronal radial migration and abnormal neuronal differentiation resulting in deficient neurite outgrowth, axon path-finding and connectivity. It is worth noting that in all these conditions, various brainstem and cerebellar abnormalities have been described, but RES has never been identified [5, 14].
Other pathophysiological mechanisms that may cause primary microcephaly have been recently identified, among others microRNAs and Golgi trafficking defects [46, 58]. But to our knowledge, microcephaly due to excessive adhesion of progenitors in the germinal zones (VZ, inner SVZ and outer SVZ) has never reported so far.
In the present case, no lesions that could argue for one of the above mentioned pathophysiological mechanisms were identified. Although lat-1, the orthologue of vertebrate ADGRLs in C. elegans, is required for cell division plane orientation in the C. elegans embryo [41, 51], normal proliferative indices in the LGE indicate that neither symmetric nor asymmetric divisions are altered by comparison with the fœtus harbouring a deleterious variant in the MCPH1 gene and in which the proliferative index was reduced by a factor of two. Also due to normal proliferative indices, extreme microcephaly with no sulcation associated with this ADGRL2 variant is unlikely to be connected to mitotic spindle dysfunction. The variation in the ADGRL2 gene is responsible for Ca2+i release alteration that constitutes the step before the modulation of microtubule organization. Furthermore, neuronal cells acquire abnormal adhesion and aggregation properties that make them stay in the subventricular zone and probably thereafter prompt them to die. All these data highlight a new mechanism for microcephaly that results from an excess of cell adhesion of neuronal progenitors in the germinal zones, which could also at least partly explain the co-occurrence of RES.
Unlike ADGRL2 variations that have never been reported to be responsible for human pathologies, variants in other adhesion-GPCR have been recognized, notably ADGRG1 (previously named GPR56), which is responsible for an autosomal recessive condition associated with the presentation of bilateral fronto-parietal polymicrogyria associated with cerebellar hypoplasia, dysgenesis and pseudo-cysts [6, 59]. Similar to humans, Adgrg1/Gpr56 null mice display a malformed cerebral cortex resembling cobblestone lissencephaly with similar cerebellar lesions . The rostral cerebellar defects result from specific failure of adhesion of the late migrating granule cells to extracellular molecules of the glia limitans whose structural integrity is disrupted with subsequent overmigration of granule cells into the subarachnoid spaces, but not from intrinsic defects in neuronal proliferation and migration . In contrast to ADGRG1/GPR56, which promotes cell adhesion of developing neurons to basal lamina molecules, ADGRL2 promotes cell migration by controlling Ca2+i release.
In conclusion, we have identified an ADGRL2 variation very likely responsible for severe microcephaly with almost no sulcation highlighting a new mechanism for the two associated malformations related to excessive adhesion of neuron progenitors within the germinal zones at least mediated by reduction of Ca2+ transients. Given its role in the determination of the anteroposterior axis suggested by its orthologue lat-1 [41, 51], we also hypothesize that RES results from abnormal positional cue alterations and defective expansion of the medial VZ in the cerebellar primordium. The identification of ADGRL2 ligands and/or of pathogenic variations in other genes associated with RES will shed more light on the role of ADGRL2 in the pathophysiology of this rare condition. Finally, in addition to its role in synapse assembly, our observations reveal the role of ADGRL2 in development, as already shown for other adhesion GPCRs.
We would like to thank Pr. Alexander Petrenko for the pcDCIRL-2 plasmid constructs and anti-CIRL-2 rat antibodies. We would also like to acknowledge Dr. Géraldine Joly-Helas and Dr. Pascal Chambon for providing control amniocyte culture cells.
Co-supported by European Union and Région Normandie. Europe is involved in Normandy through the European Regional Development Fund (ERDF).
MV performed NGS experiments and analysis and drafted the manuscript. MV and ML carried out molecular and cellular studies. MR and DV performed MRI study. VD and LR carried out in situ hybridization. LP and SO made possible the RES collection study. SC and IT belongs to NGS facilities. LT followed mother's pregnancy. HA made possible the fetuses’ collection study. TF and BG are the team directors. BG participated to the design of the study and coordination and perfomed the statistical analysis. PSV and AL conceived the study and drafted the manuscript. All authors read and approved the final manuscript.
All procedures were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
The authors declare that they have no competing interest.
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