Candidate Gene Sequencing of LHX2, HESX1, and SOX2 in a Large Schizencephaly Cohort

INTRODUCTION

Schizencephaly is an uncommon malformation of cortical development with an estimated prevalence in the United States of 1.54 per 100,000 individuals [Curry et al., ²⁰⁰⁵]. Patients with schizencephaly present with a broad range of neurological symptoms, including developmental delay, seizures, and hemiparesis or quadriparesis. Neuroimaging studies show unilateral or bilateral full thickness gray matter-lined clefts of the cerebral hemispheres. The two sides of a schizencephalic cleft can appear fused with a pial-ependymal seam (termed “closed lip”), or they can be separated by cerebrospinal fluid (“open lip”) [Yakovlev and Wadsworth, ^1946a,b]. Such clefts are most often found in the perisylvian region, and the surrounding cortex is typically polymicrogyric [Barkovich and Norman, ¹⁹⁸⁸; Barkovich and Kjos, ¹⁹⁹²; Granata et al., ¹⁹⁹⁶; Hayashi et al., ²⁰⁰²].

The suspected causes of schizencephaly are heterogeneous and remain poorly understood. Both genetic and non-genetic etiologies have been postulated [Bubis and Landau, ¹⁹⁶⁴; Komarniski et al., ¹⁹⁹⁰; Hehr et al., ²⁰¹⁰]. There are few reported associations with chromosomal aneuploidy, single gene defects, and distinct syndromes. Schizencephaly is usually simplex, but familial occurrence has been reported indicating one or more genetic factors for schizencephaly [Robinson ¹⁹⁹¹; Hosley et al., ¹⁹⁹²; Hilburger et al., ¹⁹⁹³; Tietjen et al., ²⁰⁰⁵].

A role for EMX2 was initially suggested in reports of multiplex and simplex cases [Capra et al., ¹⁹⁹⁶; Faiella et al., ¹⁹⁹⁷; Granata et al., ¹⁹⁹⁷], but more recent studies have not demonstrated a major role for EMX2 in schizencephaly [Tietjen et al., ²⁰⁰⁷; Merello et al., ²⁰⁰⁸; Hehr et al., ²⁰¹⁰]. LHX2, another gene expressed in the developing forebrain, specifies cortical fate in the hippocampus and neocortex [Hebert and Fishell, ²⁰⁰⁸] and has been hypothesized as a potential candidate gene for schizencephaly. Lhx2 knockout mice have a striking phenotype with an essentially absent neocortex [Monuki and Walsh, ²⁰⁰¹; Mangale et al., ²⁰⁰⁸].

Schizencephaly is often associated with other CNS anomalies, including hydrocephalus, arachnoid cysts, and partial or complete absence of the septum pellucidum (ASP). In fact, up to 70% of cases of schizencephaly are associated with absent or abnormal septum pellucidum, and up to one-fourth of schizencephaly cases fall into the spectrum of septo-optic dysplasia (SOD) [Barkovich and Norman, ¹⁹⁸⁸; Barkovich et al., ¹⁹⁸⁹; Barkovich, ²⁰⁰⁵]. Conversely, nearly half of the patients reported with SOD have schizencephaly [Barkovich et al., ¹⁹⁸⁹; Lau et al., ¹⁹⁹³].

The coexistence of SOD and schizencephaly suggests potential common etiologies for both phenotypes. The majority of patients with SOD are simplex, and several etiologies have been postulated, including genetic abnormalities, viral infections, and vascular events. Some multiplex cases have been reported, supporting the role of genetics underlying SOD [Benner et al., ¹⁹⁹⁰; Wales and Quarrell, ¹⁹⁹⁶; Kelberman and Dattani, ²⁰⁰⁷]. Both homozygous and heterozygous missense mutations in HESX1 have been reported in association with SOD [Dattani et al., ¹⁹⁹⁸; Thomas et al., ²⁰⁰¹]. Mutations in HESX1 have also been reported in patients with ectopic posterior pituitary gland [Brickman et al., ²⁰⁰¹] and in a patient with periventricular heterotopia and ectopic posterior pituitary gland [Mitchell et al., ²⁰⁰²]. The Hesx1 homeobox gene is expressed early in development in the mouse forebrain, and there is evidence for an important role for Hesx1/HESX1 in the development of mouse and human forebrain and pituitary gland [Dattani et al., ¹⁹⁹⁸]. Given the evidence for a role of HESX1 in SOD and the frequent association between SOD and schizencephaly, we hypothesized that HESX1 mutations might be responsible for some proportion of simplex cases of schizencephaly.

The report of a patient with SOD and schizencephaly with a heterozygous mutation in SOX2 (c.389G > C, p.G130A) [Kelberman et al., ²⁰⁰⁶] makes this another attractive candidate gene for schizencephaly. SOX2, expressed in early neuroepithelial progenitors, is a transcription factor implicated in the development of the cerebral cortex, pituitary gland, eye, and inner ear.

Despite the strong suspicion of a genetic etiology for schizencephaly, the majority of cases remain unexplained. Based on the coexistence of schizencephaly and SOD, we hypothesized that mutations in genes previously shown to be mutated in patients with SOD can also cause schizencephaly, particularly in those patients with features of SOD but perhaps also in those without SOD. Given the associations described above, we evaluated 97 cases of schizencephaly, including 13 with both schizencephaly and features of SOD, to screen for mutations in LHX2, HESX1, and SOX2.

SUBJECTS AND METHODS

RESULTS

DISCUSSION

Though schizencephaly is a heterogeneous condition, we strongly suspect that some cases have an underlying genetic etiology. Nonetheless, there are to date no genes identified that play a major role in this condition. We hypothesized that mutations in LHX2 might give rise to schizencephaly given the important role of LHX2 in cortical patterning. Because of the overlap sometimes observed between schizencephaly and SOD, we also hypothesized that genes associated with SOD—HESX1 and SOX2—might also cause some cases of schizencephaly.

We observed several polymorphisms in LHX2, including published SNPs. One LHX2 sequence variation, c.724G > A, had not been previously reported. Though it is synonymous, this does not occur in any of the functionally important LHX2 domains and is adjacent to a large known polymorphism. Thus, we concluded that this change represents a benign though uncommon polymorphism. We observed only one polymorphism in HESX1; even though it represented a non-synonymous variation in the amino acid sequence, it is a previously reported common polymorphism (http://hapmap.ncbi.nlm.nih.gov). Similarly, we observed a polymorphism in the 3′-UTR of SOX2 in a single case that we concluded was not pathogenic [Fantes et al., ²⁰⁰³; Bakrania et al., ²⁰⁰⁷].

From a total of 97 cases of schizencephaly, there were 14 with concurrent features suggestive of SOD, including ASP, optic nerve hypoplasia, and pituitary gland abnormalities. As this is a retrospective study and full records were not available from all cases, it is difficult to estimate accurately the true coincidence of SOD among all studied cases of schizencephaly. Our findings in a large but heterogeneous group of subjects with schizencephaly, a subset of whom had features of SOD, indicate that these candidate genes do not account for a significant fraction of schizencephaly or schizencephaly + SOD cases.

It is clear from familial studies that at least some occurrences of schizencephaly have a genetic etiology, but the extent to which genetic causes can explain these conditions remains unknown. The observation of schizencephaly in combination with some or all of the features of SOD in some patients may be secondary to concomitant injury to multiple structures in the developing brain. It is plausible that some mutations may cause schizencephaly by increasing the susceptibility of the developing brain to environmental events. Additional studies will be needed to better define the genetic and non-genetic factors that contribute to schizencephaly.