TLE1, a key player in neurogenesis, a new candidate gene for autosomal recessive postnatal microcephaly
Introduction
A large number of neurodevelopmental disorders are characterized by a normal head size at birth followed by a deceleration in head growth, resulting in postnatal microcephaly with an occipital frontal circumference (OFC) less than 3 standard deviations (-3SD) below the mean. These disorders, also referred to as « postnatal microcephalies », occur particularly when the white matter is affected, during infancy and/or childhood (Passemard et al., 2017).
Subsequent to the classic recognizable syndromes, such as Rett and Angelman syndrome, and those more recently described such as CDKL5 or FOXG1, Rubinstein-Taybi, and Pitt-Hopkins syndromes, Next Generation Sequencing (NGS) has facilitated the identification of new emerging phenotypic entities. Many postnatal microcephaly syndromes are caused by mutations in genes known to be important in the regulation of gene expression in the developing forebrain and hindbrain, although important synaptic structural genes also play a role (Seltzer and Paciorkowski, 2014).
Among these, FOXG1 syndrome is characterized by postnatal microcephaly, severe global developmental delay and cognitive impairment beginning early in infancy. These children generally do not attain ambulation and have dyskinetic movement disorders, feeding difficulties, and occasional Rett-like features (Ariani et al., 2008; Dastidar et al., 2012; Kortüm et al., 2011; Mitter et al., 2017; Papa et al., 2008; Shoichet et al., 2005). Imaging of patients with FOXG1 syndrome typically reveals comprised foreshortened frontal lobes, delayed myelination, reduced white matter volume and corpus callosum anomalies (Bahi-Buisson et al., 2010; Kortüm et al., 2011).
Here, we report on a homozygous missense mutation in the TLE1 gene, encoding for a transcriptional repressor interacting with FOXG1. The patient presented postnatal microcephaly with severe developmental delay, and, on MRI, progressive cortical atrophy, delayed myelination and thin corpus callosum, which was suggestive of a FOXG1-like presentation.
The female patient was the third-born child of healthy first cousins from Pakistan. Her elder brother and sister were healthy and there was no family history of developmental delay or congenital anomaly syndromes. Routine ultrasonography during pregnancy was normal. She was born at 39 weeks of gestation by normal vaginal delivery, with weight 2870 g (-1 SD for age and gender), length 47 cm (-1 SD) and head circumference 32 cm (-2 SD). The early neonatal course was normal.
At the age of one month, she developed intermittent episodes of crying with peripheral and axial dyskinesia. Subsequently, head growth started decelerating at the age of 3 months, leading to progressive microcephaly. At the age of 6 months, her development was profoundly delayed, since she could not smile or hold her head. On examination, orofacial dyskinesia was prominent, leading to a failure to thrive and a request for an enteral nutrition. She also demonstrated extreme truncal hypotonia, intermittent opisthotonic posture and peripheral hypertonia. Standard ophthalmic examination revealed no eye tracking, no light reaction but normal fundus oculi.
At last examination, at 6 years of age, growth parameters were in the normal range: weight 18.5 kg (M) and height 105 cm (M) but the microcephaly was still worsening, head circumference 43 cm (-6 SD). She had made virtually no progress, she had no head control, she showed severe axial hypotonia associated with spastic quadriplegia, leading to orthopaedic complications including scoliosis and hip dislocation. No epileptic seizures were reported but she experienced intermittent axial and peripheral non-epileptic myoclonus. She had no eye contact, and no ability to communicate. She also had significant swallowing and feeding difficulties with orofacial dyskinesia, therefore requiring a gastrostomy tube placement.
Repeated MRI, performed at the ages of 6 and 22 months, showed cortical progressive atrophy with myelination delay and thin but complete corpus callosum (Fig. 1). CT scan did not show calcification. Exhaustive metabolic screening including blood lactate and pyruvate, plasma and urine amino acids, urine organic acids, CSF lactate, CSF/plasma glycine ratio, VLCFA, CMV, were all normal. Echocardiography was normal. Repeated EEG showed a disorganized pattern characterized by excessive diffuse slow activity with right posterior spikes. Electrophysiological investigations, including Visual Evoked Potential, electroretinogram, auditive evoked potential, electromyogram and peripheral nerve conduction studies, were normal.
Subsequent to the clinical and the imaging presentation, the patient phenotype was described as « FoxG1-like syndrome”.
To identify the underlying cause of the phenotype trio based exome sequencing was performed. A homozygous substitution in TLE1 (NM_001300303.1), c.1651G > A, was identified, leading to the missense variant p.Asp541Asn. Bioinformatics analysis indicated that the mutated amino acid is highly conserved across species (phyloP 7.76) from humans to C. elegans. The mutation is predicted to be deleterious (SIFT score: 0; median 3.71) and disease causing (Mutation Taster p-value: 1). The variant is present in dbSNP (rs201140985), NHLBI, Exome Variant Server (EVS), the Exome Sequencing Project (ESP) and Exome Aggregation Consortium (ExAC) Browser, but with an extremely low occurrence (MAF = 0,0001), and is absent from in-house NGS analyses. No other potentially pathogenic variations were identified in other genes. The presence of the mutation in the patient DNA and autosomal-recessive segregation in all available family members was confirmed by bidirectional Sanger sequencing (supplementary figure).
In order to search for additional mutations, Sanger sequencing of the 20 exons of TLE1 was performed in a replicative cohort of 10 patients from consanguineous families showing FOXG1-like features, but no other variants were identified.
In order to determine the functional consequences of TLE1 mutations in cell proliferation and survival, we performed immunostaining using Ki67 and pH3 (phospho-histone 3) to assess the proliferative and mitotic indices, respectively, in dermal fibroblasts obtained from a skin biopsy of the patient and sex/age-matched controls. We found a significant decrease in the mitotic and proliferative indices in patient cells compared to control (Fig. 2). Altogether, our data suggest that TLE1 mutation lead to a lengthening of cell cycle and aborted cell division.
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Discussion
We report here the first homozygous missense mutation in the TLE1 gene in a patient with postnatal microcephaly and FOXG1-like features. Groucho/transducin-like Enhancer of split (Gro/TLE1) is a nuclear factor that lacks intrinsic DNA-binding activity but can interact with a variety of DNA-binding proteins and acts as a transcriptional co-repressor. The TLE1 protein consists of five conserved domains: Q (glutamine-rich domain), GP (glycine/proline-rich domain), CcN (phosphorylation sites for
Funding sources
Research reported in this publication was supported by the Agence Nationale de la Recherche [ANR-16-CE16-0011 MC, AB, NBB]; the Fondation Maladies Rares; and DESIRE [grant agreement 602531]; and COST Action Proposal OC-2016-1-20862 " European Network on Brain Malformations ".
Acknowledgements
We would like to thank the patients and their families for their contribution to our research. We acknowledge Eva Coppola, Frederic Causeret, Alessandra Pierani for their fruitful discussions and contributions to this research.
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These 2 authors contribute equally to the manuscript.