Background
It is widely accepted that neutral drift and Darwinian positive selection have played an important role in the evolution of human features. During the last few years, research has been focused on human genome-wide scans of adaptative evolving loci to search for specific modern characteristics in this species [
1]. Although most of them are related to fitness, it has been reported that some genes under positive selection in the human lineage can also confer vulnerability to some diseases [
2‐
4].
Schizophrenia, which is considered as a disease related to the origin of
Homo sapiens, could be a by-product of an adaptative process [
3,
5,
6]. Previous reports have indicated a relationship between positively selected genes and schizophrenia. Crespi et al. [
3] found signals of positive selection in 28 of 76 schizophrenia candidate genes that had been previously reported as positive results in association studies. Evidence of recent positive selection in the human lineage has also been found in haplotypes of
MAOB and
GABRB2 genes, which also confer an increased risk to schizophrenia [
2,
4]. Furthermore, brain areas that are differentially dysregulated in schizophrenia include the regions most-notably subject to differential evolutionary change along the human lineage [
7‐
9]. In addition, it has recently been suggested that metabolic processes altered in schizophrenia evolved at a higher rate in the human lineage, when compared with the chimpanzee [
10].
A selective advantage could affect the achievement of specific human capacities, such as language. In this context, TJ Crow [
9,
11], postulates that schizophrenia is the price that
Homo sapiens had to pay for the acquisition of language. Moreover, recent neuroimaging studies report impairment in brain function relevant to language processing in individuals with schizophrenia and in those who are at a genetic risk for this disease [
12].
First evidence for a gene involved in language was reported in 2001, when the
FOXP2 gene was identified by Lai et al. [
13]. Identification of the transcriptional targets of FOXP2 revealed that this protein could regulate genes involved in development and function of the brain, genes under positive selection in human lineage and genes associated to schizophrenia [
14]. Apart from the polyglutamine tracts, the human protein only differs in three amino acids from its ortholog in mouse, and two of these changes occurred in the human lineage after separation from the common ancestor shared with chimpanzees. Both changes are fixed in human populations, and there is evidence to support they have been under positive selection [
15,
16].
Association studies between
FOXP2 polymorphisms and susceptibility to different pathologies of language impairment, such as specific language impairment, dyslexia or autism have not produced robust results [
17], but the identification of two coding mutations related to verbal dyspraxia [
18]. Nevertheless there are strong evidence of the importance of the gene in development and some aspects of language [
19] including the fact that CNTNAP2, a downstream target of FOXP2 has been related also to language disorders [
20,
21]. In schizophrenia, preliminary association studies have delivered controversial results [
22‐
24]. To the best of our knowledge, no methylation study of
FOXP2 has previously been done.
We hypothesized that FOXP2 could be considered a candidate gene that may confer vulnerability to schizophrenia or to the language related symptoms of this disorder. To test this hypothesis, two different analyses were carried out: 1) an association study between FOXP2 polymorphisms and schizophrenia and 2) the study of the methylation status of the FOXP2 promoter in different areas of the brain in patients and controls.
Methods
Association study participants
For the association study, 293 patients and 340 healthy unrelated controls were analyzed. All patients and controls were Caucasians of Spanish descent. Exclusion criteria included organic brain syndromes, mental retardation, severe drug abuse, or inability to understand simple questions. Participants with previous psychiatric treatment were excluded as controls.
There were no significant differences in sex or age for both groups. All patients met DSM-IV criteria for schizophrenia. The Manchester scale [
25], and the psychotic symptom rating scale (PSYRATS) [
26], were used respectively, to assess the clinical psychotic symptoms, with particular attention to the Poverty of speech item, and the intensity of auditory hallucinations. The mean Manchester score was 8.79 (SD = 5.56) and mean PSYRATS score was 16.26 (SD = 23.26). This study was approved by the local Ethics Committee. All patients signed the informed consent form.
Post-mortem human brain samples
For methylation and expression analyses, human brain samples were kindly donated by the London Neurodegenerative Diseases Brain Bank at the Institute of Psychiatry. Grey tissue from both hemispheres of the superior temporal gyrus, parahippocampus gyrus and cingulate gyrus was obtained. For methylation analyses, one sample for each region was analyzed for both patients and controls. For expression analyses, 13 samples from patients (6 from the right hemisphere and 7 from the left hemisphere) and 12 samples from controls (9 from the right and 3 from the left hemisphere) were analyzed.
Association study
Genomic DNA was extracted from peripheral blood leukocytes by the Puregene kit (Gentra Systems, MN, USA).
A total of 27 polymorphisms were analyzed, 10 of them by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and 17 by an iPLEX genotyping assay (Sequenom, CA, USA). Details of the primer sequences, PCR conditions and restriction enzymes are described in Additional files
1 and
2.
Three regions were screened for potential trinucleotide expansions: two polyQ tracts of 40 and 10 residues, located respectively in exons 5 and 6 of
FOXP2, and a CGG-rich region in intron s1 close to the transcription start site. Primers flanking the three regions were designed (see Additional file
1). One primer in each pair was 5'-labeled with 6-FAM or HEX fluorophores. Fluorescent amplicons were electrophoresed with internal lane size standards in an ABI PRISM
® 3700 DNA Analyzer (Applied Biosystems Inc.) and length of fragments was analyzed with the GeneScan-v3.7 (Applied Biosystems, Inc.).
Statistical and genetic analyses were performed using Haploview v4.1, UNPHASED 3.10, and SSPS v13 software. Bonferroni correction was used for multiple tests. For the haplotype association study, four marker sliding-windows were used, with the exception of a five marker haplotype, for which association had been detected in a previous study [
24].
Methylation analysis
DNA from brain samples was extracted using a Nucleon® Genomic DNA Extraction Kit (Tepnel Life Sciences). DNA from leukocyte samples was extracted using the Puregene kit (Gentra Systems).
DNA was fragmented with EcoRI (New England Biolabs) prior to overnight digestion with Proteinase K (Sigma Aldrich). DNA was cleaned, purified and concentrated using a Qiaex II kit (Qiagen). The processed DNA samples were treated with either the CpGenome™ DNA Modification Kit (Chemicon® International) or the EpiTect Bisulfite Kit (Qiagen) in accordance with the supplier's guidelines.
DNA was amplified with specific primers for bisulphite-converted DNA (see Additional file
1). PCR fragments were cloned into the PCR 2.1 vector using the TOPO cloning kit (Invitrogen), or pGEM-T
® vector using the pGEM-T
® Easy Vector System (Promega), and sequenced with T7 and SP6 universal primers.
Expression analysis
Total RNA was extracted with the RNeasy Lipid Tissue Mini Kit (Qiagen). Reverse transcription of 1 μg of RNA was performed using SuperScriptTM III Reverse Transcriptase (Invitrogen) and random primer hexanucleotides (Promega).
Quantitative RT-PCR was performed in triplicate for each sample on an iCycler iQ Real Time PCR System (Qiagen) with Power SYBR
® Green PCR Master Mix (Applied Biosystems) using a standard protocol. Specific cDNA primers for
FOXP2 and
RPII, used as a control gene, were designed. Sequences and PCR conditions are shown in Additional file
1. The comparative CT method (ΔΔCT) was used to measure the relative gene expression.
Discussion
In this study we investigated the role of FOXP2, a positively selected gene, in schizophrenia vulnerability. A SNP association study, with particular attention to language related symptoms as auditory hallucinations and poverty of speech, and a study of DNA methylation and expression of this gene were carried out.
The most important finding of this study is the significant association showed between the rs2253478 SNP and the item of Poverty of speech of the Manchester scale (p = 0.038 after Bonferroni correction). This polymorphism is located in intron s3, not close to any of the promoter regions. There is no information that it could be an enhancer of splicing element. Its potential functionality has not been yet investigated, and then it is difficult to determine the biological significance of this association. Alternatively it could be in linkage disequilibrium with another polymorphism being the causative factor. In any case, our results relate the
FOXP2 gene to one of the characteristic symptoms of schizophrenia, deficits in the language domain [
27‐
29].
On the other hand, the haplotypic analysis confirmed our previous results that the rs7803667T/rs10447760C/rs923875A/rs2396722C/rs2396753A haplotype could be a protective one with respect to auditory hallucinations [
24].
It has been suggested that specific language-related circuits are affected in patients with schizophrenia [
12]. Therefore, it is reasonable to look for risk alleles to schizophrenia vulnerability in
FOXP2, a gene for which an implication in the development of language is well accepted [
13,
30]. Nevertheless, schizophrenia is a biological entity not well defined, indicative of a phenotype too much complex for genetic analysis, which partially explains the difficulty to find the causative genetic factors. At this point, the study of language variables in order to find risk alleles in schizophrenia becomes a good alternative with respect to endophenotype approaches. Our results support this hypothesis, since significant results were found when we related language impairment in schizophrenic patients to
FOXP2 polymorphisms.
In this work, we also analyzed whether the polyQ stretches at exons 5 and 6 of
FOXP2 are polymorphic and if so, determine its potential association with schizophrenia vulnerability. Expansions in the number of trinucleotides repeats are frequently associated with neurodegenerative diseases [
31]. However, no variation in the number of glutamines was found in our sample. This high stability is concordant with previous studies in controls, individuals with progressive movement disorders, and schizophrenic patients [
32,
33]. The role of the polyQ tracts in the
FOXP2 gene is unknown. In fact, most of the members of the FOX family lack this domain. Nevertheless, the high invariability of these sequences suggests that they could be under functional constraints.
In addition to schizophrenia vulnerability due to variations in the DNA sequence, epigenetic factors regulating gene expression have also been suggested as a potential etiological mechanism in psychosis [
34,
35]. Epigenetic regulation has been increasingly associated with psychiatric disorders, with examples in depression and addiction [
36,
37].
In our study of DNA methylation of
FOXP2 exon s1 region, we found a higher degree of methylation in the left hemisphere of the parahippocampus gyrus region in patients than in controls. From these results, we would have expected lower gene expression of
FOXP2 due to repression by methylation. However, no differences were found in
FOXP2 expression between controls and patients. This discrepancy could be explained by the fact that only a stretch of the CpG island, located in exon s1, was analyzed for methylation. The promoter region of the
FOXP2 gene has not been well defined, and regulation of the gene is more complex than was initially thought (non published personal data, [
38]). The finding that expression data show a trend of more expression in patients than in controls would indicate that a decrease of neural processes controlled by the protein FOXP2, a repressor of transcription, is produced in patients. Hippocampal and parahippocampal volume reduction is one of the most consistent findings in schizophrenia [
39]. Moreover, in a meta-analysis of brain volumes in relatives of patients with schizophrenia, hippocampal reduction was the largest difference between relatives and healthy controls [
40]. These findings suggest hippocampal volume as a potential end of phenotype for genetic studies in schizophrenia.
Our study has some limitations. First, the language skills evaluated in this work include only two items of the Manchester scale. Since the strongest result is related to one of these items, we would recommend a systematic exploration of language variables in schizophrenic patients. Therefore, it would be valuable to explore different aspects of language in future studies. Second, we have used a small sample in the methylation and expression analyses so further studies with a larger sample would be necessary in order to confirm our preliminary results. Finally, other variables which could affect methylation, such as medication or age, should be considered. In spite of these limitations, this study suggests the use of the language related disorder as alternative phenotypes in schizophrenia for genetic studies. On the other hand, although the results are not conclusive, this is the first epigenetic study of FOXP2 in schizophrenia, opening a new way in which this gene could be related to this disorder.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AT participated in the experimental procedure, analysis of results and the draft of the manuscript. AMD participated in the experimental procedure and the draft of the manuscript. MDM, and JS participated in the conception and the design of the study. RF conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors contributed to and have approved the final manuscript.