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Correspondence and requests for materials should be addressed to Anne-Sophie Denommé-Pichon, Unité Fonctionnelle Innovation en Diagnostic génomique des maladies rares, FHU-TRANSLAD, CHU Dijon Bourgogne, 15 boulevard Maréchal De Lattre de Tassigny, 21070 Dijon CEDEX, France
Functional Unit for Diagnostic Innovation in Rare Diseases, FHU-TRANSLAD, Dijon Bourgogne University Hospital, Dijon, FranceUMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, FranceEuropean Reference Network, ERN-ITHACA
Functional Unit for Diagnostic Innovation in Rare Diseases, FHU-TRANSLAD, Dijon Bourgogne University Hospital, Dijon, FranceUMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, France
UMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, FranceDepartment of Neurology, Dijon Bourgogne University Hospital, Dijon, France
Functional Unit for Diagnostic Innovation in Rare Diseases, FHU-TRANSLAD, Dijon Bourgogne University Hospital, Dijon, FranceUMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, France
UMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, FranceDepartment of Genetics and Reference Center for Development Disorders and Intellectual Disabilities, FHU-TRANSLAD and GIMI Institute, Dijon Bourgogne University Hospital, Dijon, France
Functional Unit for Diagnostic Innovation in Rare Diseases, FHU-TRANSLAD, Dijon Bourgogne University Hospital, Dijon, FranceUMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, France
European Reference Network, ERN-ITHACAInstitute of Medical and Molecular Genetics (INGEMM), La Paz University Hospital, Autonomous University of Madrid, IdiPAZ, Madrid, SpainRare Diseases Networking Biomedical Research Centre (CIBERER), Carlos III Institute, Madrid, Spain
European Reference Network, ERN-ITHACAInstitute of Medical and Molecular Genetics (INGEMM), La Paz University Hospital, Autonomous University of Madrid, IdiPAZ, Madrid, SpainRare Diseases Networking Biomedical Research Centre (CIBERER), Carlos III Institute, Madrid, Spain
Genetics Department, Poitiers University Hospital, Poitiers, France; University of Poitiers, Poitiers, FranceExperimental and Clinical Neurosciences Laboratory, INSERM, University of Poitiers, Poitiers, France
Medical Genetics, Azienda Ospedaliero-Universitaria Senese, Siena, ItalyMedical Genetics, University of Siena, Siena, ItalyMed Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Siena, Italy
Department of Molecular and Developmental Medicine, University of Siena, Siena, ItalyU.O.C. Pediatria, Azienda Ospedaliera Universitaria Senese, Siena, Italy
Functional Unit for Diagnostic Innovation in Rare Diseases, FHU-TRANSLAD, Dijon Bourgogne University Hospital, Dijon, FranceUMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, FranceEuropean Reference Network, ERN-ITHACA
Functional Unit for Diagnostic Innovation in Rare Diseases, FHU-TRANSLAD, Dijon Bourgogne University Hospital, Dijon, FranceUMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, France
Functional Unit for Diagnostic Innovation in Rare Diseases, FHU-TRANSLAD, Dijon Bourgogne University Hospital, Dijon, FranceUMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, FranceDepartment of Genetics and Reference Center for Development Disorders and Intellectual Disabilities, FHU-TRANSLAD and GIMI Institute, Dijon Bourgogne University Hospital, Dijon, France
Functional Unit for Diagnostic Innovation in Rare Diseases, FHU-TRANSLAD, Dijon Bourgogne University Hospital, Dijon, FranceUMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, France
UMR1231 GAD “Génétique des Anomalies du Développement”, INSERM, FHU-TRANSLAD, University of Burgundy, Dijon, FranceEuropean Reference Network, ERN-ITHACADepartment of Genetics and Reference Center for Development Disorders and Intellectual Disabilities, FHU-TRANSLAD and GIMI Institute, Dijon Bourgogne University Hospital, Dijon, France
Miller-Dieker syndrome is caused by a multiple gene deletion, including PAFAH1B1 and YWHAE. Although deletion of PAFAH1B1 causes lissencephaly unambiguously, deletion of YWHAE alone has not clearly been linked to a human disorder.
Methods
Cases with YWHAE variants were collected through international data sharing networks. To address the specific impact of YWHAE loss of function, we phenotyped a mouse knockout of Ywhae.
Results
We report a series of 10 individuals with heterozygous loss-of-function YWHAE variants (3 single-nucleotide variants and 7 deletions <1 Mb encompassing YWHAE but not PAFAH1B1), including 8 new cases and 2 follow-ups, added with 5 cases (copy number variants) from literature review. Although, until now, only 1 intragenic deletion has been described in YWHAE, we report 4 new variants specifically in YWHAE (3 splice variants and 1 intragenic deletion). The most frequent manifestations are developmental delay, delayed speech, seizures, and brain malformations, including corpus callosum hypoplasia, delayed myelination, and ventricular dilatation. Individuals with variants affecting YWHAE alone have milder features than those with larger deletions. Neuroanatomical studies in Ywhae−/− mice revealed brain structural defects, including thin cerebral cortex, corpus callosum dysgenesis, and hydrocephalus paralleling those seen in humans.
Conclusion
This study further demonstrates that YWHAE loss-of-function variants cause a neurodevelopmental disease with brain abnormalities.
Miller-Dieker syndrome (MDS; [MIM 247200]) is a contiguous gene deletion syndrome caused by a microdeletion involving 26 coding genes on human chromosome 17p13.3, including both PAFAH1B1 and YWHAE.
MDS is characterized by neurodevelopmental delay with classical lissencephaly type 1, microcephaly, brain abnormalities, seizures, and craniofacial abnormalities including a narrow and furrowing forehead, low-set and posteriorly rotated ears, small eyes, upslanting palpebral fissures, epicanthus, a small nose with low nasal bridge, upturned nares, thin upper lip, cleft palate, and small chin. Additional manifestations include cardiac malformations, urogenital abnormalities, and a failure to thrive. Life span is drastically shortened, with few patients surviving past early childhood.
Smaller deletions or loss-of-function variants involving the proximal PAFAH1B1 gene alone (formerly named LIS1 [MIM ∗601545]) are responsible for isolated lissencephaly (LIS1; [MIM 607432]),
Refinement of a 400-kb critical region allows genotypic differentiation between isolated lissencephaly, Miller-Dieker syndrome, and other phenotypes secondary to deletions of 17p13.3.
Am J Hum Genet.2003; 72 (http://doi.org/10.1086/374320): 918-930
making PAFAH1B1 the main determinant for lissencephaly in patients with MDS. Although the PAFAH1B1-associated clinical features are well documented, the specific contribution of the YWHAE gene to MDS is understudied. Apart from case report with only one case with a small 12.6-kb intragenic deletion of the YWHAE gene presenting brain abnormalities,
the link between YWHAE variants alone and human disorders remains understudied, showing a need to build a cohort of individuals with YWHAE variants alone.
The YWHAE gene (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon isoform [MIM ∗605066]) encodes the 14-3-3 epsilon protein. 14-3-3ε is highly conserved and implicated in intracellular events involving phosphorylation-dependent switching or protein-protein interactions.
The auditory brainstem response shows that mouse Ywhae is also involved in hearing and mutant mice have increased minimum detection thresholds at all frequencies.
Here, we report and analyze a series of 10 patients, along with 5 cases previously published, with heterozygous loss-of-function YWHAE variants collected through international collaborations. The effect of the YWHAE loss of function is further investigated through phenotypic characterization of a homozygous knockout mouse model we generated at the Wellcome Sanger Institute (United Kingdom). Together, our results establish a new neurodevelopmental disease caused by loss-of-function YWHAE variants both in humans and mice.
Materials and Methods
Human
Patient recruitment
Written informed consent for study participation and publication was obtained by the attending geneticist or the referring physician from the patients’ legal guardians.
Collection of cases: Data sharing networks
Cases were collected through data sharing networks. The cases included in this study were gathered through GeneMatcher,
the Decipher database, the ITHACA European Reference Network, the French AnDDI-Rares network, and through personal correspondence. We included cases with single-nucleotide variants (SNVs) in YWHAE or deletions of less than 1 Mb encompassing YWHAE but not PAFAH1B1. We chose not to include cases with PAFAH1B1 deletion to increase detection power of phenotypes specific to YWHAE.
Clinical characteristics were assessed through a standardized phenotyping sheet and included data on magnetic resonance imaging–based neuroimaging.
Genetic testing and targeted RNA study
In all patients, variants and deletions disrupting YWHAE were detected by either chromosomal microarray analysis or next-generation sequencing. For a description of chromosomal microarray analysis and next-generation sequencing methodology, see the Supplemental Methods. All variants reported in this study refer to YWHAE transcript NM_006761.5. Genomic coordinates refer to the GRCh37 human reference genome. SNVs were classified using American College of Medical Genetics and Genomics guidelines.
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
For the cases with SNVs, we set out to assess the variant’s impact on splicing. Cases 2 and 3 (2 monozygotic twins carrying the same variant) underwent targeted RNA study. Total RNA was extracted from whole blood collected in a PAXgene tube (Preanalytics GmbH, Hombrechtikon, Switzerland) using the PAXgene Blood RNA kit (Preanalytics GmbH, Hombrechtikon, Switzerland) following the standard protocol. Complementary DNA was obtained using the QuantiTect Reverse Transcription kit (Qiagen GmbH, Hilden, Germany). RNA from case 1 was not available and has not been studied. The conditions for reverse transcription polymerase chain reaction (RT-PCR) are detailed in the Supplemental Methods.
Phenotypic features comparison
Patients for whom a specific feature information was missing were not included in that feature’s statistic; hence, the group sizes differ between features. We used the Fenton preterm growth charts with term-corrected age to assess birth measurements and World Health Organization growth charts from 0 to 5 years and from 1 to 18 years to assess measurements after birth.
Mouse
Generation of Ywhae knockout mouse model
The knockout mouse model was generated by homologous recombination using the knockout-first allele method, producing the Ywhaetm1e(EUCOMM)Wtsi knockout allele. The Ywhae gene was targeted in C57BL/6N embryonic stem cells with a conditional-ready cassette (clone EPD0200_5_G01) as described previously.
Germline transmission was confirmed by a series of genotyping and quantitative polymerase chain reaction analyses using the standard International Mouse Phenotyping Consortium quality control validation strategy, with the results found here (https://www.mousephenotype.org/data/genes/MGI:894689). Mice derived from heterozygous (Het) intercross were genotyped for the Ywhaetm1e allele by polymerase chain reaction carried out as previously described.
The line was then crossed with 129S5/SvEvBrd/Wtsi to improve viability. Mice were produced from Het × Het matings to give segregating litters that included homozygous knockout (Hom), Het, and WT as littermate controls. A subset of these mice was used in neuroanatomical characterization.
Mouse whole-body phenotyping studies
The Ywhaetm1e(EUCOMM)Wtsi knockout mice were phenotyped for whole-body traits by the Mouse Genetics Project pipeline at the Wellcome Sanger Institute, United Kingdom (for details, see the Supplemental Methods). In brief, mice of both sexes were weighed between 4 and 16 weeks of age and assessed at 9 weeks for gross behavioral abnormalities (modified SHIRPA
) and dysmorphology using standardized procedures. For each test, Ywhaetm1e/tm1e were tested alongside littermate Ywhae+/+ controls through the pipeline, which included tests for indirect calorimetry, body composition, x-ray morphology, blood chemistry, and hematology (n = 6 for WT and n = 7 for Hom mice).
Comprehensive neuroanatomical studies
Neuroanatomical studies were carried out using 3 Ywhaetm1e/tm1e male and 5 WT male mice (2 littermates and 3 mice from the same mixed background) at 16 weeks of age. Mice were anesthetized with ketamine (100 mg/kg, intraperitoneal) and xylazine (10 mg/kg, intraperitoneal). Brains were dissected and fixed in 4% neutral buffered formalin for 48 hours, then transferred to 70% ethanol. Samples were embedded in paraffin using an automated embedding machine (Sakura Tissue-Tek VIP) and cut at a thickness of 5 μm with a sliding microtome (Leica RM 2145) to obtain coronal brain region at Bregma +0.98 mm according to the Allen Mouse Brain Atlas. The sections were then stained with 0.1% Luxol Fast Blue (Solvent Blue 38; Sigma-Aldrich) and 0.1% cresyl violet acetate (Sigma-Aldrich) and scanned using Nanozoomer 2.0HT, C9600 series at 20× resolution, as previously described.
Twenty-two brain parameters, made up of area and length measurements, were taken blind to the genotype. Using in-house ImageJ plugins, an image analysis pipeline was used to standardize measurements. Each image was quality controlled for the accuracy of sectioning relative to the reference Allen Mouse Brain Atlas and controlled for asymmetries and histological artifacts.
At Bregma +0.98 mm, the brain structures assessed were as follows: (1) the total brain area, (2) the lateral ventricles, (3) the cingulate cortex, (4) the genu of the corpus callosum, (5) the caudate putamen, (6) the anterior commissure, (7) the piriform cortex, (8) the primary motor cortex, and (9) the secondary somatosensory cortex. All samples were also systematically assessed for cellular ectopia (misplaced neurons). Mouse neuroanatomical data were analyzed using a two-tailed t test assuming equal variance.
Mouse embryogenesis and high-resolution episcopic microscopy
To study mouse embryogenesis, we generated Het × Het timed matings on a mixed C57BL/6N and 129S5/SvEvBrd/Wtsi genetic background. We produced 44 embryos at embryonic day 14.5 (E14.5), of which 3 Hom and 3 WT were sent to the Deciphering the Mechanisms of Developmental Disorders consortium (https://dmdd.org.uk) for high-resolution episcopic microscopy (HREM) processing. The detailed protocol has been published previously.
High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology.
Briefly, 14.5-day embryos were embedded in a hard plastic resin, enabling thin sectioning (1 μm). This resin contains a fluorescent dye, which allows contrast between the tissue and the bright background of the plastic. By sequentially imaging the block face during the sectioning process, a comprehensive stack of accurately aligned images was acquired, documenting the 3-dimensional structure of the sample. We then carried out a comprehensive assessment of brain morphometrics by adapting our 2-dimensional histology protocol used in adult mice to 3-dimensional brain stacks.
Microdeletions including YWHAE in the Miller-Dieker syndrome region on chromosome 17p13.3 result in facial dysmorphisms, growth restriction, and cognitive impairment.
J Med Genet.2009; 46 (http://doi.org/10.1136/jmg.2009.067637): 825-833
In total, 15 patients (7 males, 7 females, and 1 unknown) from 14 unrelated families with heterozygous loss-of-function YWHAE variants were collected: 3 splice site variants (cases 1-3), 2 intragenic deletions (cases 10 and 14), and 10 large deletions (<1 Mb), encompassing YWHAE but not PAHAFAB1 (cases 4-9, 11-13, and 15) (Figure 1A and B ).
Figure 1YWHAE variants reported in this study and analysis of splice site variant. A. YWHAE variants at the gene level shown using the UCSC Genome Browser tool
(GRCh37 assembly): single-nucleotide variants in cases 1, 2, and 3 and deletions <1 Mb encompassing YWHAE in cases 4 to 15. Five cases present with variants specifically in YWHAE, including 3 splice variants and 2 intragenic deletions. Genes highlighted in green are reported in OMIM to be disease causing, whereas genes in gray are not disease causing. YWHAE location is depicted in cyan. Coding exonic regions are depicted with gray shading. B. Variants in cases 1, 2, and 3 are positioned at the protein level (NM_006761.5 NP_006752.1). Coding exon regions and 14-3-3 domain are depicted with gray shading. C. Left panel: analysis by gel electrophoresis of exons 2 to 6 RT-PCR products from cases 2 and 3 showing 2 bands corresponding to 2 RNAs of different sizes. Right panel: study of variant impact on premessenger RNA splicing using targeted RNA deep sequencing showing heterozygous exon 5 skipping. cDNA, complementary DNA; RT-PCR, reverse transcription polymerase chain reaction.
Variants occurred de novo (10 of 12) or were inherited from a symptomatic parent (1 of 12) or from a healthy father with paternal mosaicism (1 of 12). In 3 of 15 cases, inheritance is not known.
The c.715+1G>T variant present in cases 2 and 3 affects the donor splice site of intron 5 of YWHAE. Analysis of RT-PCR products from cases 2 and 3 by gel electrophoresis showed 2 bands corresponding to 2 RNAs of different sizes (Figure 1C). The study of variant impact on premessenger RNA splicing using targeted complementary DNA sequencing showed the skipping of exon 5 (NM_006761.4:r.579_715del) in a heterozygous state, with 40,920 of 87,290 reads in case 2 and 10,437 of 22,352 reads in case 3, leading to the deletion of 127 nucleotides, predicted to cause a frameshift and create a premature stop codon p.(Leu194∗) (Figure 1C). These additional results confirm that the c.715+1G>T variant results in an aberrant splicing of the YWHAE gene.
The main clinical features are summarized in Table 1 and fully detailed in Table 2. The mean age at the time of the last evaluation was 10 years (from 19 weeks of gestation to 39 years old). There was no apparent difference in severity between males and females. Almost all patients had developmental delay (13 of 14) with impaired speech or language (11 of 11). The age of the first sentences ranged from 2 years 6 months to 3 years 4 months (mean of 2 years 7 months). Motor delay was less common: 3 of 10 started to walk after 18 months (from before 12 to 30 months), 3 of 8 had impaired gait, and 4 of 7 impaired hand skills such as dyspraxia (2 of 7). When present and evaluable, intellectual disability (ID) (6 of 13) was mild to severe, and, interestingly, variants strictly within YWHAE were associated with ID in only 1 case (case 1), presenting with mild ID. Behavioral disorders were frequent (6 of 12), and some patients were reported with attention-deficit/hyperactivity disorder (3 of 12) and anxiety (3 of 12). Growth retardation was observed only in patients with a deletion encompassing more than just YWHAE (4 of 11). Hypotonia seemed to be frequent (7 of 12), accompanied by feeding difficulties in 2 patients. Tremor was reported (2 of 11). Seizures were frequent (8 of 13) with a mean age of onset of 2.4 years (from 4.5 months to 9 years). Seizures were tonic-clonic, myoclonic, spasms, absence, or febrile. Some individuals had several types of seizure, and 2 of 8 patients presented with multidaily seizures. Brain malformations were common (8 of 12) (Figure 2A) and included signs of altered myelination (ventricular dilation [3 of 8], corpus callosum hypoplasia [5 of 8], poor myelination [3 of 8]), cysts (subependymal germinolytic, septum pellucidum, cavum vergae, arachnoid, and near optic nerve) (4 of 8), cerebellar hypoplasia (3 of 8), enlarged Virchow-Robin spaces (3 of 8), thinning of the frontal cortex (2 of 8), absence of the olfactory bulbs (2 of 8), Chiari malformation type 1 (1 of 8), intraventricular hemorrhage (1 of 8), pituitary hypoplasia (1 of 8), polymicrogyria (1 of 8), and nodular heterotopias (1 of 8). Craniofacial features were observed in 12 of 15 patients, among which 3 of 5 with variants strictly within YWHAE, presenting milder and nonspecific dysmorphic features (Figure 2B). Skeletal defects were observed in 7 of 13 patients, with clinodactyly or camptodactyly of the fourth or the fifth fingers in 4 of 7 individuals. Although individuals did not have their hearing tested, no impairments were reported.
Table 1Comparative summary of clinical features between individuals with variants in YWHAE only, individuals with variants in YWHAE and other genes, and Ywhaetm1e/tm1e mice, from this study and a literature review
The severity of the signs increases with the number of “+” symbols. The symbol +/− indicates inconsistent results between mouse studies, which we briefly comment on here: for the assessment of anxiety, Ikeda et al
; therefore, it could be a false positive. To facilitate the interpretation of the murine findings, we also provide the full nomenclature of the models used in the literature review: for Toyo-oka et al,
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
Strong diffuse multifocal paroxysmal activity (spike-and-wake and polyspike-and-wave)
NA
NA
Disorganized sleep and wake patterns, rather diffuse abnormalities during sleep
Irregular spike-wave complexes on the right side
Infantile spasms, wake and sleep differentiated at the diagnosis of infantile spasms and at the last follow-up the EEG was normal
NA
NA
NA
NA
NA
NA
Brain MRI or CT
Pituitary hypoplasia, small unilateral arachnoid cyst
Intraventricular hemorrhage, germinolytic cyst, ventricular dilatation, absence of the olfactory bulbs, hypoplasia of the cerebral white matter, thin corpus callosum, enlarged Virchow-Robin spaces, cerebellar vermis hypoplasia
Ventricular dilatation, cyst of the septum pellucidum and the cavum vergae, thin corpus callosum and enlarged Virchow-Robin spaces, delayed myelination, cerebellar vermis hypoplasia, absence of the olfactory bulbs
Slight widening of anterior portion of left sylvian fissure, reduced volume of inferior frontal parenchyma with widening of ipsilateral superficial liquor spaces, poor myelination
Nodular heterotopias, hypoplasia of the corpus callosum, polymicrogyria, abnormal bulging of the brainstem
Normal
Normal
NA
Normal (thalamic restriction: vigabatrin?)
NA
Not performed
Thinning of corpus callosum and frontal cortex
Colobomatous cyst near right optic nerve, normal aspect of the white matter and enlarged Virchow-Robin spaces
Chiari I, thin corpus callosum, thin cavum septum pellucidum and cavum vergae
NA
Behavioral disorders
No
Anxiety, skin scratching, ADHD, short attention span
Anxiety, skin scratching, ADHD, short attention span
Figure 2Brain MRIs and photographs in patients. A. Brain MRI images of cases 1 (at 6 months old), 2, and 3 (at 3 years old). (a-c) T1-weighted sagittal brain MRI. (a) Case 1: pituitary hypoplasia (arrow). (b) Case 2: ventricular dilatation, enlarged Virchow-Robin spaces (arrowheads), germinolytic cysts (star), and thin corpus callosum. (c) Case 3: ventricular dilatation, enlarged Virchow-Robin spaces (arrowheads), and thin corpus callosum. (d-j) T1-weighted axial brain MRI. (d) Case 2: enlarged Virchow-Robin spaces (arrowheads). (e) Case 3: enlarged Virchow-Robin spaces (arrowheads). (f) Case 2: ventricular dilatation and germinolytic cysts (star). (g) Case 3: ventricular dilatation and septum pellucidum cyst (cross). (h) Case 3: ventricular dilatation and cavum vergae cyst (arrow). (i) Case 2: cerebellar vermis hypoplasia (arrow). (j) Case 3: cerebellar vermis hypoplasia (arrow). (k) T2 weighted coronal brain MRI in case 1: presence of the olfactory bulbs. (l) T2 Fast recovery fast spin echo weighted coronal brain MRI in case 2: absence of the olfactory bulbs. (m) T2 Fast spin echo weighted coronal brain MRI in case 3: absence of the olfactory bulbs. (n-o) T2 Fluid-attenuated inversion recovery weighted axial brain MRI. (n) Case 2: isointense white matter, with loss of gray/white dedifferentiation and hypersignal of the posterior white matter showing delayed myelination with age (arrow). (o) Case 3: isointense white matter, with loss of gray/white dedifferentiation showing delayed myelination with age, ventricular dilatation, septum pellucidum (cross), and cavum vergae cysts (arrow). B. Photographs of cases 4 and 10 from top to bottom. MRI, magnetic resonance imaging.
We next thought to characterize and evaluate datasets from a Ywhaetm1e(EUCOMM)Wtsi knockout mouse model with a focus on the most frequent clinical components of the human disorder. Results of knockout mice from this study and a literature review are summarized in Table 1.
According to the literature, phenotypes were more severe in Ywhae−/− mutants when compared with Ywhae+/− mice. With respect to the 3R (replacement, reduction, and refinement) principles to limit animal use, heterozygous Ywhaetm1e mutant mice were not studied because our main goal was to gain insights into the in vivo consequences of YWHAE full inactivation. In our previous research, Ywhaetm1e/tm1e mice showed reduced body weight from weaning age at all time points until 16 weeks of age (Figure 3A adapted from
). The area under the curve was 22% lower in Ywhaetm1e/tm1e compared with WT animals (P < .05, t test). At 12 weeks, these differences were mostly driven by a reduction in body length and lean body mass (−8%, P < 5 × 10−5 and −15%, P < 5 × 10−3, respectively) (Figure 3B and C). To determine if these growth delays might also occur during embryogenesis, we assessed embryo morphology in a group of 13 homozygous embryos of the 44 collected. We found that 23% (3 of 13) exhibited embryonic growth retardation, 38.5% (5 of 13) presented with a fetal edema, and 31% (4 of 13) displayed a pale yolk sack (data not shown), suggesting that growth delays originate from embryonic stages.
Figure 3Ywhaetm1e/tm1emice display an array of developmental and neuroanatomical phenotypes. A. Body weight in Ywhaetm1e/tm1e male mice from 4 to 16 weeks (data from Ingham et al
). B, C. Body length and mean mass. D. Cumulative food consumption over 21 hours measured in calorimetric cages. E. Fat mass at 14 weeks. F-H. Bone area, mineral content, and density (14 weeks). I. Histogram with fold increase/decrease expressed as percentage of wild-type mice. Plain lines regroup parameters or left and right hemispheres. Dotted lines indicate left and right hemispheres. Colored regions indicate the presence of at least one significant parameter within the brain region at the 0.05 level. J. Schematic representation of affected brain regions in mice plotted in coronal planes (Bregma +0.98 mm) according to P values. The color map indicates P value below the threshold of .05 or gray in which the n was too low to calculate statistics. K. Nissl-stained coronal brain sections from WT and Ywhaetm1e/tm1e mice, showing the reduction of the cingulate cortex, the reduction of the width of the genu of the corpus callosum, the enlargement of the lateral ventricles, and an unusual mass of axons beneath the genu of the corpus callosum at the midline (black arrow). L. HREM analysis of 14.5 embryos showing on the left, enlarged ventricle (parasagittal sections) and on the right panel, the thinning of the cortical plate and subventricular layer cortex (coronal sections). The vertical blue lines on the parasagittal section (left) indicate the corresponding coronal plane (shown on right panel), whereas the horizontal blue lines indicate the corresponding longitudinal plane (not shown) using ImageJ plugins. Aca, anterior commissure; cc, corpus callosum; Cg, cingulate cortex; CPu, caudate putamen; CxP, cortical plate; gcc, genu of the corpus callosum; HREM, high-resolution episcopic microscopy; LV, lateral ventricle; M1, primary motor cortex; Pir, piriform cortex; S2, second somatosensory cortex; SubVCx, subventricular layer cortex; TBA, total brain area; WT, wild-type.
Food consumption was measured over 21 hours in metabolic cages, and Ywhaetm1e/tm1e mice displayed a reduced latency to feeding initiation (Figure 3D). Water intake, however, was normal (data not shown). After 5 hours, the cumulative food intake resumed with similar increments compared with WT, highlighting that the earlier increase in food intake could be the result of hyperactivity, which has been reported before.
Thus, the trend toward increase in food consumption does not mirror fat mass (Figure 3E) or fat percentage (24.4% ± 2.5 vs 21% ± 3.2 in WT and Ywhaetm1e/tm1e, respectively), which were identical in both genotypes. Instead, body length and lean mass were significantly reduced (−8%, P < 10−4 and −16%, P < .005, respectively), highlighting growth delay. Indirect calorimetric analysis in individual cages highlighted hyperactivity at night (Supplemental Figure 1 and Supplemental Table 1). The respiratory exchange ratio was slightly elevated in Ywhaetm1e/tm1e mice, indicative of greater use of carbohydrates as a substrate, and could again be correlated to hyperactivity because this feature was only present for low respiratory exchange ratio readings and only during the first 5 hours of the acclimatization period (Supplemental Figure 1). This suggests that physical alterations in Ywhaetm1e/tm1e mice result from developmental abnormalities rather than a metabolic origin. Standard measures of plasma metabolites showed an increase in sodium (+2%), glycerol (+48%), alkaline phosphatase (+41%), and aspartate aminotransferase (+42%) (Supplemental Table 2). Hematology profiles showed increased white and red blood cell counts (+48% and +12%, respectively) (Supplemental Table 3). Bone area and mineral content showed a reduction (−19.2%, P < 10−6 and −22.9%, P < 10−4, respectively), whereas bone density was not affected (not significant) (Figure 3F-H). Except for a shortened skull and a concave nasal spline, the bone shape and morphology assessed by radiography (Supplemental Table 4) and other variables, such as coat and skin color, head size and morphology, fore- and hindlimb size and morphology, digit fusion, and nail counts, were normal in Ywhaetm1e/tm1e mice.
A previous study reported an abnormal morphology of the hippocampus when Ywhae is inactivated in the mouse.
In this study, comprehensive neuroanatomical phenotyping of adult and embryos in Ywhaetm1e/tm1e mice allowed us to find new features relevant to human YWHAE variants that have not been reported before.
Among the 22 brain parameters assessed at Bregma +0.98 mm, severe and multiple brain defects, including microcephaly, were identified in male Ywhaetm1e/tm1e mice and are summarized in Figure 3I and J. The total brain area at Bregma +0.98 mm was reduced (−18%, P = .02). Strikingly, mutant mice showed a compression of the genu of the corpus callosum width (−67%, P = 10−5) associated with enlarged lateral ventricles (+394% on average, P < 10−4) (Figure 3K). In addition, we observed an unusual mass of axons ventrally contiguous to the midline of the corpus callosum (black arrow, Figure 3K) that could potentially originate from dysregulation in the navigation of postcrossing axons in the corpus callosum as previously described.
Fronto-medio-dorsal cortical areas were also reduced in size with the cingulate cortex area, width, and height all being significantly affected (P < .05). Interestingly, white matter structures were also reduced in size independently of local pressure from other parameters. This was the case for the anterior commissure (−43%, P < 10−4), which is situated on the floor of the caudate putamen (not significant). Basic neurologic assessment did not reveal any gross abnormalities (Supplemental Table 5), although not every neurologic function was tested because it has been extensively published elsewhere.
To discriminate congenital brain malformations from acquired brain abnormalities (when the brain is normal at birth but shows abnormalities subsequently), we studied brain morphology at embryonic day 14.5 (E14.5) using HREM datasets (Figure 3L). More specifically, we reassessed phenotypes present in adults using the murine developmental brain atlas
and online software available through the Deciphering the Mechanisms of Developmental Disorders consortium website. Lateral ventricle enlargement was clearly visible on parasagittal planes, whereas thinning of cortical layers (cortical plate and cortical subventricular layer) was also evident on coronal sections (black arrows, Figure 3L). Together, these results indicate that neuroanatomical defects pertaining to ventricles, white matter structures, and brain size originate early during embryonic life.
Discussion
Here, we report clinical manifestations of 15 patients with clear dose-dependent loss-of-function variants in YWHAE, 5 of which are patients with variants affecting only YWHAE showing developmental and speech delays (5 of 5) as well as brain malformations (4 of 4), establishing YWHAE as a gene causing a rare neurodevelopmental disease. Splice variants were carried by 3 of the 15 patients, whereas 12 of 15 had deletions encompassing the YWHAE gene (2 small intragenic deletions predicted to cause a frameshift and 10 larger deletions encompassing additional genes in 17p13.3). When the inheritance was known, variants occurred de novo with the exception of 2 variants inherited from a symptomatic parent or a healthy mosaic father.
We did not identify any recurrent variant—except for the one present in the 2 monozygotic twins—nor did we identify pathogenic nonsense, missense, or indel variants in YWHAE. Some regions of YWHAE present a strong constraint to missense variants because they do not seem to tolerate any such variant (gnomAD v2.1.1).
Although we only identified loss-of-function variants in YWHAE, we do not exclude the possibility that missense variants could also alter 14-3-3ε protein function. It is noteworthy that the Decipher (ID 359873 and 288974) and ClinVar (VCV000148756 and VCV000154956) databases show 4 additional deletions of YWHAE alone, one of which (VCV000154956) is located in coding regions, reported in an individual with autistic behavior. No further clinical details and inheritance information are provided on these online databases, although autism is known to be associated with disorders of the corpus callosum.
We identified 4 additional individuals with YWHAE variants alone bringing the total number of patients with specific variants in YWHAE to 5. This allowed a better delineation of the core phenotypes associated specifically with YWHAE loss of function, which consistently included global developmental delay with impaired language, hypotonia, and brain malformations, including corpus callosum hypoplasia, delayed myelination, and ventricular dilatation (see Table 1 for a summary or Table 2 for a detailed phenotypic description). We found notable neuroanatomical phenotypic overlap with homozygous Ywhaetm1e/tm1e mutant mice, which displayed multiple brain structure defects, including hydrocephaly, dysgenesis of the corpus callosum, small anterior commissure, and thin motor and cingulate cortices, present before birth. The novelty of our murine findings in the context of previous Ywhae mouse reports
allowing us to detect more subtle neuroanatomical differences (see Figure 3K for an example).
Seizures and behavioral abnormalities were commonly present in patients (see Table 1). Dysmorphic facial features seem to be milder when compared with individuals with deletion encompassing more than just YWHAE. Only 1 of 5 patients presented with mild ID. Cases with a variant specifically in YWHAE did not show growth retardation in contrast to patients with more than 1 gene affected, which included the CRK gene previously reported to be associated with growth retardation in the MDS.
Microdeletions including YWHAE in the Miller-Dieker syndrome region on chromosome 17p13.3 result in facial dysmorphisms, growth restriction, and cognitive impairment.
J Med Genet.2009; 46 (http://doi.org/10.1136/jmg.2009.067637): 825-833
were seen in our mice and epilepsy was not modeled. However, in a previous study dedicated to neurocognitive behaviors in Ywhae homozygous knockouts, mice showed increased locomotor activity and deficits in cognition.
Although the precise molecular role of 14-3-3ε in the development of the corpus callosum remains unclear, it has been shown that 14-3-3 proteins regulate the switch from sonic hedgehog–mediated commissural axon attraction to repulsion after midline crossing in the developing mouse spinal cord.
The involvement of 14-3-3ε in a switch that controls repulsive guidance clues was further evidenced in motor axons derived from the fruit fly CNS model system.
It is thus tempting to speculate that the mass of axons we detected beneath the corpus callosum (black arrow, Figure 3K) may be due to misregulation of commissural axon attraction and repulsion, potentially caused by the absence of 14-3-3ε. Through its interactions with LIS1 and NUDEL, 14-3-3ε is also known to interact with dynein. When 14-3-3ε is deficient in mice, there is a reduction in cytoplasmic dynein function, thereby underlining its role in neuronal migration.
Via its interactions with dynein, it could be hypothesized that loss of 14-3-3ε could affect corticogenesis and explain the thinning of the motor and cingulate cortices in mice. However, further analyses should be performed to study the precise function of 14-3-3ε protein in corticogenesis and colossal axon crossing at the midline.
Conclusion
To our knowledge, this study presents the first molecular and clinical description of a rare neurodevelopmental disorder occurring with brain abnormalities caused by heterozygous loss-of-function SNVs in YWHAE and gathers new or previously reported cases with deletion encompassing YWHAE but not PAFAH1B1. Moreover, studies of Ywhaetm1e/tm1e mice showed neuroanatomical defects similar to those seen in the human condition, reinforcing the importance of YWHAE in the proper development of the mammalian brain.
Together, these results suggest that YWHAE loss of function results in a neurodevelopmental syndrome with brain abnormalities involving corpus callosum dysgenesis with milder phenotypes than the contiguous gene deletion syndrome of chromosome 17p13.3 without the PAFAH1B1 deletion.
To improve the identification and management of this new syndrome, the clinical data would need to be enriched, in particular with respect to the neurocognitive profile and the long-term outcome of these patients.
Data Availability
The datasets generated during this study are available upon request from the corresponding authors.
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgments
We thank the families for taking part in the study. We thank the CCuB for technical support and management of the computing platform. We thank ERN-ITHACA and the GeneMatcher platform for data sharing. Several authors are members of ERN-ITHACA. We acknowledge Christophe Ouzouf, Jérémie Roquet, Mathieu Chopelet, and Laura Fenlon for discussion or providing comments on the manuscript. We thank the members of the Sanger Institute Mouse Pipelines teams and the Research Support Facility for the provision and management of the Ywhaetm1e/tm1e knockout mice.
Funding
This work was supported by grants from the French National Institute of Health and Medical Research (First Step to BY), the French National Research Agency (ANR JCJC to BY), the European Union through the FEDER program (to GAD), and by the Wellcome Trust Grant 206194. The Deciphering the Mechanisms of Developmental Disorders (DMDD) program was funded by the Wellcome Trust (Grant 100160) with support from the Francis Crick Institute, licensed under a Creative Commons Attribution license (CC BY 4.0). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
This study conforms to the Helsinki Declaration of ethical principles for medical research involving human subjects. When additional analyses not included in the care approach were required (RNA studies), they were done in the framework of the DISCOVERY project, which was approved by the appropriate French independent ethics committee (Comité de Protection des Personnes 2016-A01347-44), and samples were part of the GAD collection DC2011-1332. The care and use of mice were in accordance with the UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986. Permission was obtained to publish photographs of cases 4 and 10.
Refinement of a 400-kb critical region allows genotypic differentiation between isolated lissencephaly, Miller-Dieker syndrome, and other phenotypes secondary to deletions of 17p13.3.
Am J Hum Genet.2003; 72 (http://doi.org/10.1086/374320): 918-930
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology.
Microdeletions including YWHAE in the Miller-Dieker syndrome region on chromosome 17p13.3 result in facial dysmorphisms, growth restriction, and cognitive impairment.
J Med Genet.2009; 46 (http://doi.org/10.1136/jmg.2009.067637): 825-833
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