Autism spectrum disorder is a complex, heterogeneous, behaviorally-defined disorder with a 4:1 male:female gender distortion 1. Although environmental elements, such as peri- and post-natal stress, have been reported to contribute to the development of autism, monozygotic twin studies along with evidence of chromosomal abnormalities, mutations in single genes, and multiple gene polymorphisms in autistic individuals, clearly show that autism is a largely genetic disorder 2345.

Single mutations in neuroligin 3 and 4, cell adhesion molecules present at the post-synaptic side of the synapse, and in SHANK3, a scaffolding protein found in excitatory synapses, have been described in autistic individuals 67. In the majority of cases, however, an overall lack of Mendelian inheritance suggests the involvement of multiple genes 58. Indeed, genome-wide screens and candidate gene approaches have identified a number of chromosomal regions and genes linked with autism 910111213141516171819. For example, a strong association between autism and SLC25A12, a gene encoding the mitochondrial aspartate/glutamate carrier AGC1 expressed in neurons and in neural stem cells, has been reported in 2 separate studies 1719. Similarly, an analysis of chromosome 16p revealed an association between autism and the protein kinase c-beta gene (PRKCB1), which is expressed in granule cells of the brain and B lymphocytes 20.

Although most of the genetic analyses, to date, have focused on genes expressed in the brain, the pathophysiology of autism suggests that other systems such as the immune system and the pituitary-hypothalamic axis may be involved 2122. In some autistic individuals, for example, abnormal secretion of pro-opio-melanocortin (POMC), adrenocorticotropin (ACTH), cortisol, and beta-endorphin has been noted 22232425.

We recently performed a genome-wide linkage scan in a group of families with autism and phrase speech delay identified in the Autism Genetic Resource Exchange (AGRE) DNA repository 20. Among the 7 genomic regions that showed a significant increase in identity-by-descent sharing in sibling pairs with autism, 4 corresponded to regions that have previously been linked to autism (chromosome 5, 13, 16, and 17) 20. Based on these results, in the current study, we focused on chromosome 5q31 and identified 3 genes that we hypothesized could be involved in the development of autism: 1) paired-like homeodomain transcription factor 1 (PITX1), which is a key regulator of hormones within the pituitary-hypothalamic axis such as ACTH, cortisol, and beta-endorphin 222627; 2) histone family member H2AFY, which is involved in X-inactivation in females and therefore could be a positional candidate that could explain the 4:1 male:female gender distortion present in autism 128; and 3) Neurogenin 1 (NEUROG1), which is a transcription factor involved in neurogenesis 29. Using single point association analyses and haplotype analyses, we found significant evidence for an association of autism with PITX1 but not with H2AFY or NEUROG1.

In this study, we have found a significant association between autism and polymorphisms of PITX1, a paired-like homeodomain transcription factor involved in hormonal regulation 2627. Using a two step procedure we initially identified evidence for association for marker rs3805663 with autism. In the second step additional markers in the PITX1 gene were genotyped in an extended sample set of 276 families total. Although in this extended set marker rs3805663 did not reach a significant p-value any more, several additional markers showed highly significant results with the most significant result for rs6596189 (p = 1 * 10^-4). Haplotype analyses yielded a couple of highly significant pairings, although none of these showed higher significance than rs6596189 alone. Individuals homozygous or heterozygous for the risk allele were 2.69 and 1.74 fold more likely to be autistic than individuals who were not carrying the allele, respectively.

PITX1 is a key regulator of hormonal genes in the pituitary-hypothalamic axis. Its putative involvement in autism is supported by evidence documenting abnormal levels of hormones such as ACTH, beta-endorphin, and cortisol in autistic individuals and by the fact that these hormones are downstream of PITX1 222324252627. Deregulation of POMC and high levels of beta-endorphin in the morning, for example, have been shown to be involved in certain maladaptive behaviors, such as self-injurious behaviors, which are often seen in autistic individuals 37. The ACTH-cortisol system, which also plays an important role in stress related responses, is impaired in autistic individuals in whom lower cortisol levels and higher ACTH levels have been reported 24.

Linkage between chromosome 5q31-32 and autism is consistent with previously published studies. Both the IMGSAC (1998) genome-wide linkage scan and the screen performed by Risch and colleagues found modestly elevated LOD-scores in this region and exclusion mapping analyses did not clearly exclude this region 1012.

Interestingly, this genomic region was recently identified as a potential locus for attention deficit/hyperactivity disorder (ADHD) 383940. In a genome-wide scan using large multi-generational pedigrees, Arcos-Burgos and colleagues established an exclusion map for the region and defined a critical interval from 119 to 135 Mb, which encompasses the PITX1 gene region 38. Their most significant family-specific microsatellite marker D5S2117 at 133.4 Mb is less than 1 Mb from the PITX1 gene locus.

Mutation screening of the coding region of PITX1 revealed a single known mis-sense mutation with a MAF of 0.33. As the single point association results did not show any evidence for association, the mutation seems unlikely to be directly involved in susceptibility to autism. The most positive SNPs in the single marker analysis as well as the positive haplotypes are all situated in the first intron of the PITX1 gene. Regulatory site analysis shows a prediction for an alternative promoter at bases 134394831–134395400, which overlaps exactly with the region covered by the four most significant SNPs. However, to our knowledge activity of this predicted promoter has not been shown. Also these SNPs are in strong LD with SNPs within the 5' region of the PITX1 gene and the designated promoter region. However, one SNP within the PITX1 promoter, rs7700313 genotyped by us did not show significant evidence for association (p_nominal = 0.0578, Table 3). Therefore at this stage it is not possible to postulate where the functional variant is situated and if any of the SNPs genotyped is functional.

The linkage region also covers the H2AFY and NEUROG1 loci, which in view of their function in X-chromosome inactivation and neuronal development, respectively, were considered reasonable candidates 28294041. We found associations neither for the 5 SNPs selected for H2AFY nor for the 2 SNPs selected for NEUROG1. Moreover, mutation screening by direct sequencing did not show any amino-acid or other mutational changes within these genes.

Although the mechanisms by which PITX1 may contribute to the susceptibility to autism are yet to be explored, the genetic association between PITX1 polymorphisms and autism, described here, could provide an explanation for the abnormal level of hormones of the pituitary-adrenal axis reported in the literature.

All authors are salaried employees of Integragen SA.

ER, JH, and PB were responsible for developing study concepts and design. FR, KF and CV were responsible for laboratory work such as genotyping. AP, FT, JC and ML performed the statistical analyses. AB, PG, and PL were responsible for the bioinformatics. The manuscript was approved by the authors.