The engrailed homeobox protein (en) plays an important role during development in the invertebrate segmentation, where it is required for the formation of posterior compartments in larva segments. In vertebrates, the EN protein has been involved the regionalization of the mid-hindbrain segment of the neural tube. Indeed, it is a reference transcription factor coding positional information in the cerebellum morphogenesis and retinotectal synaptogenesis (Martinez et al., 1991; Friedman and O’Leary, 1996; Cheng et al., 2010). Alexandra Joyner research group described the En1 and En2 homeobox-containing paralog genes in vertebrates and their expression during embryogenesis (Joyner et al., 1985; Joyner and Martinz, 1986). Then, Logan et al. (1989, 1992) mapped human EN1 to chromosome 2 and EN2 to chromosome 7.

In the mouse, the En2 expression in the mid-hindbrain segment is induced by Fgf8 signal from the isthmic organizer (Crossley et al., 1996; Martinez et al., 1999) and it is required to specify mesencephalic and cerebellar development, including serotoninergic and dopaminergic neurons in the raphe nuclei, substantia nigra, and ventral tegmental area (Millen et al., 1994; Wurst et al., 1994; Simon et al., 2005; Sonnier et al., 2007). In chick embryos, the expression gradient of En2 in the tectum regulates the normal establishment of the retinotopic pattern of retinal innervation (Araki and Nakamura, 1999; Nakamura and Sugiyama, 2004).

The relationship between the autistic spectrum disorder (ASD) and EN2 gene polymorphisms and mutations has been suggested since 1995 (Petit et al., 1995). More recently genome-wide association studies showed a potential role of EN2 in ASD (Benayed et al., 2005, 2009). Moreover, the EN2 abnormal expression and its methylation profile have been reported in the cerebellum of ASD patients (James et al., 2013, 2014; Choi et al., 2012, 2014; Jamuar et al., 2014). The mechanisms underplaying ASD related to EN2 expression anomalies are still controversial (Yang et al., 2008, 2010). For instance, A-C specific polymorphism in EN2 gene (A-C in rs1861972 (A/G) and rs1861973 (C/T)) has been associated to EN2 over-expression in ASD individuals. Conversely, in Yang et al. (2010) A-C appears as a protective haplotype against ASD in Han Chinese ethnic group (see also Chien et al., 2011). The minor G-T haplotype is over-represented in unaffected siblings (Gharani et al., 2004; Benayed et al., 2005). The EN2 overexpression may be a potential causal mechanism of developmental alterations in brain morphogenesis, generating a deregulated growth between the cerebellum and cortex (Rubenstein, 2010). This deregulation may represent an alteration in the spatial and temporal pattern of synaptogenesis that finally could determine behavioral phenotypes associated to ASD. Human brain postmortem studies and neuroimaging analysis have reported different degrees of cerebellar atrophy and cortical anomalies of dopaminergic/serotoninergic innervation in ASD brains (for review see, Palmen et al., 2004). Actually, Skefos et al. (2014) found that the mean overall Purkinje cell density was lower in the cases with autism as compared to controls. These structural alterations may be explained by abnormal development of the mid-hindbrain neural tube segment. In addition, brain anomalies due to the EN2 haploinsufficiency have been also proposed as a cause of mental retardation in 7q terminal deletion syndrome (Frints et al., 1998; Linhares et al., 2013). Although these experimental and clinical data support the importance of the EN2 protein function in brain development, to date no studies have been published to try to establish an association between clinical and cerebral structural phenotypes and abnormalities in EN2 gene expression.

Therefore, we have studied the phenotypical characteristics of a series of patients with encephalic structural anomalies (ESA) and who showed abnormalities in the DNA and/or transcribed RNAm of EN2 (EN2-g) and compare them with patients with: (1) ESA and other gene mutations (OG-g), including: PAX2 and FGF8, key factors for neural regionalization (Hidalgo-Sánchez et al., 2005), LIS1 (also known as PAFAH1B1) and functionally related genes, involved in neuronal proliferation and migration in the developing brain (Spalice et al., 2009; Guerrini and Parrini, 2010); and (2) ESA and patients free of these type of mutations (NM-g), showing all of them similar clinical conditions.

The engrailed homeobox protein (EN) distribution in the mid-hindbrain segment is fundamental to understand the development and function of the isthmic organizer that regulates the cerebellum and midbrain morphogenesis (Figure 2). At neural tube stages, the isthmic organizer signal, Fgf8, determine the central band of high level of EN expression at the isthmus, and the bilateral decreasing expression of EN over mesencephalic and rhombencephalic neuroepithelium (Crossley et al., 1996; Martinez, 2001). This gradiental distribution of EN protein has been demonstrated as a mechanism of positional information required for topographic organization of synapsis in the retinotectal system (Nakamura and Sugiyama, 2004). In addition, EN expression is required to specify serotoninergic and dopaminergic neurons in the hindbrain, which control cerebral cortex function trough raphe-cortical and meso-cortical projections, respectively. Therefore, alterations of EN expression might be associated to synaptic anomalies underlying neurodevelopmental disease in humans. Actually, genetic alterations in the expression of EN2 have been related to different neurologic conditions and more particularly to autism spectrum disorder (ASD).

ASD is a neurodevelopmental disorder with a strong genetic component. A close relationship between ASD and EN2 specific polymorphisms, as well as genetic and epigenetic alterations, has been established in several studies, suggesting that the etiologic influence of EN2 variants in ASD may be explained through genetic mechanisms (Petit et al., 1995; Blair et al., 2002; Brune et al., 2008; Benayed et al., 2009; Sen et al., 2010; Viaggi et al., 2015). Although the underlying genetic architecture is not well understood, two theories explain the genetic roots of common complex diseases, including the ASD. The first is the common variant-common disease hypothesis (CVCD), which suggest that ASD is due to a combination of frequent genetic allelic variations (>1%), each conferring a modest risk (odds ration < 1.5) (Bodmer and Bonilla, 2008). The second one is the rare variant-common disease (RVCD) hypothesis, in which the genetic risk of ASD is explained by rare mutations conferring a significant risk (Talkowski et al., 2014). Since the RVCD is favored by recent studies on genetic causes of ASD (Huguet and Bourgeron, 2016; Liu and Takumi, 2014), we have studied in the present work the potential contribution of rare gene variants in EN2 to ASD phenotype. The analysis of EN2 gene alterations and the presence of ASD phenotypes did not support a significant relation between EN2 and ASD in our selected population of patients showing encephalic structural anomalies (ESA). We have demonstrated that EN2 gene alterations contribute to phenotypes of ESA, but they are not more related to ASD than other genetic anomalies. Nevertheless, 8 of 12 EN2-g patients have exclusively mutations in EN2 and 6 of them showed ASD of different degrees: Cases 1, 10, and 12 showed PD; Case 5 showed nuclear autism, Cases 7 and 8 showed mild autism, and Cases 4 and 2 did not show autism; suggesting a potential contribution of EN2-related developmental processes in autism physiopathology. But, the lacking of massive genomic and transcriptomic studies did not allow us to eliminate a pathogenic influence of other genetic alterations. In addition, we have not studied EN2 expression in ASD patients without ESA, where pathogenic influence of EN2 protein expression may become evident.

Regarding the possibility that the ethnic difference might confound the EN2 association with autism, five out of twelve EN2-g patients have three different ethnical backgrounds apart from Caucasians (three Maghrebian, one Romany, and one South American). Although the sample is not large enough to be conclusive, it could be suggestive of ethnic association between EN2 gene alterations and brain anomalies in groups with higher traditional consanguinity.

The main feature associated to the reported EN2 abnormalities in this study, is MR, but this was similarly expressed among OG-g patients who did not show these EN2 abnormalities. However, MR was far less frequent among those patients of NM-g. There are few series studies reporting EN2 and MR association (Laroche et al., 2008), although it seems to be universally spread among single case reports (Sarnat et al., 2002; Titomanlio et al., 2005). In our study, as mentioned before, all the EN2-g patients suffered from some intellectual disability, ranging from severe to mild MR, in agreement to the described phenotype in 7q terminal deletion syndrome where EN2 gene is affected (Frints et al., 1998; Linhares et al., 2013). Moreover, axonal guidance in target territories to establish adequate synaptic patterning depend of engrailed proteins concentration (Marie and Blagburn, 2003); then, decreasing of EN2 transcription could represent a haploinsufficient phenotype that predispose to MR and ASD in 7q terminal deletion syndrome. Moreover, recent findings En2 knockout mice display reduced levels of tyrosine hydroxylase, noradrenaline, and serotonin in the hippocampus and cerebral cortex, similar to those observed in human brains with ASD (Viaggi et al., 2015). In addition, Soltani et al. (2017), have reported an effect of En2 protein in neuronal morphogenesis and synaptogenesis of hippocampal neurons in culture. Therefore, although other associate genetic anomalies have been detected in EN2-g patients that may explain the ESA phenotypes, EN2-related alterations in cortical development and neuronal differentiation might be related to the presence MR in all of EN2-g patients. In addition, similar alterations in LIS1 related genes have been demonstrated in patients suffering schizophrenia and bipolar disorder (Tabares-Seisdedos et al., 2006), in favor of common pathogenic mechanisms underplaying neurodevelopmental disorders.

In our study, EP was not more frequent among EN2-g patients than it was in the rest of the OG-g series (75 and 70%, respectively). However, it is more frequent than in NM-g (37%). There were scarce literature published about the linkage between the EN2 mutations and epilepsy, although it may be expected because its theoretical relations with ASD (Tuchman et al., 2010). However, in mice models, Tripathi et al. (2009) found that Hippocampal En2 mRNA content decreased after seizures induced by kainic acid (KA). This suggests that En2 might also influence the functioning of forebrain areas during adulthood and in response to seizures (Tripathi et al., 2009; Tripathi and Bozzi, 2015). Moreover, our EN2-g, show in four cases, transcriptional errors in LIS1, HLIS1, HLIS3, and HLIS5, markers of LIS1[PAFAH1B1] gene, which are also present in the OG-g patients, and have been related to a wide variety of phenotypic abnormalities associated to EP (Ross, 2002).

Only four of the EN2-g patients showed gross motor impairments resembling CP, which has not been described before. Three out of four, showed also mutations in the critical region of lissencephaly (LIS1 markers; see Table 5) that can explain the macroscopic structural abnormalities associated to migration anomalies and cortical dysplasia, as has been reported in animal models and patients (Wurst et al., 1994; Yang et al., 2008; Cheng et al., 2010; Hadjivassiliou et al., 2010).

Cerebellar malformations were expected more frequent in EN2-g, attending to previous studies (Millen et al., 1994; Wurst et al., 1994; Blair et al., 2002; Spalice et al., 2009; Viaggi et al., 2015), but differences were not detected among our groups. The En1 and En2 genes have been proved sharing co-expression domains in the developing cerebellum and mesencephalon of mice (Millen et al., 1994; Cheng et al., 2010; Wilson et al., 2011). Although important anomalies have been related with down regulation of En1 and En2 knockouts in mouse mutants (Millen et al., 1994; Wurst et al., 1994; Cheng et al., 2010; Wilson et al., 2011) the possibility of mutual compensatory activity has been also proved (Hanks et al., 1995).The absence of systematic and significant cerebellar anomalies in humans with genetic alterations in EN2 may be consequence of this compensatory effect in the heterozygous background.

Neurological comorbidity rate was similar among EN2-g and OG-g patients and was less frequent among NM-g patients. Our findings suggest that the dysfunctions of genes evaluated in this study, which regulate the structural development of the CNS, induce a wide range of clinical pictures that involve most of the functional areas of the brain, resulting in a small group of neurological syndromes clinically recognizable. This would suggest that the clinical picture that they show is the product of a suboptimal brain function due to the miss organization of the brain structures. In practice, from a clinical point of view, these patients can be resumed in a group with abnormal adaptive conditions of the directive functions of the CNS, which can be more or less evident depending on the epigenetic and environmental factors and induce the similar suboptimal cognitive patterns, even with heterogeneous genetic backgrounds.

Our basic gene and cDNA EN2 screening is a preliminary study about genomic changes that are producing consequences in the transcriptomic profile. Furthermore, experiments using modern sequentiation approaches (e.g., deep sequencing and RNAseq) and in larger patient’s population will be necessary to confirm and define precisely En2 and other gene alterations in congenit brain structural anomalies.

In conclusion, EN2 gene variations analyzed in peripheral blood samples, altogether with the other genes analyzed in the present study, showed a high prevalence of unspecific clinical pictures, including ASD, as well as a major mutual comorbidity. We have not detected clear differences in the prevalence of behavioral disturbances, especially ASD between patients showing EN2 anomalies and patients with other genetic anomalies. This unspecific pattern supports the polygenic nature of ASD comprising the novo and rare inherited variants acting within the context of rare-variant genetic load (revised in Chaste et al., 2017).

Clinical data are related to a descriptive observational study on 12 patients showing EN2 mutations (Group: EN2-g), out of 109 patients coming from a referral pediatric population (under fifteen years old) of 33.291 patients, was carried out with the approval of the ethics. The reference code ethical approval to use human samples (including blood cells) is: 2016/VSC/PEA/00091 (Responsible person of Ethical Committee: Alberto Pastor; Responsible person of the Project: Salvador Martinez).

FC-M conceived and designed the work, analyzed and interpreted the clinical data, drafted the work, revised the manuscript, and agreed to be accountable for all aspects of the work, ensuring its scientific accuracy and integrity. PA-L analyzed and interpreted the clinical data. TE-M and AB-L generated and interpreted the genetic data. MM-M generated and interpreted the genetic and cellular data, and revised the draft. CB designed, generated, and interpreted the cellular and genetic data, revised the draft. SM conceived and designed the work, interpreted the genetic data, revised the draft. PA-L, TE-M, AB-L, MM-M, CB, and SM agreed to be accountable for all aspects of the work, ensuring the accuracy and integrity of the data. All authors approved the final version of the manuscript to be published.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.