Candidate gene study of HOXB1 in autism spectrum disorder

<p>Abstract</p> <p>Background</p> <p><it>HOXB1 </it>plays a major role in brainstem morphogenesis and could partly determine the cranial circumference in conjunction with <it>HOXA1</it>. In our sample, <it>HOXA1 </it>alleles significantly influence head growth rates both in autistic patients and in population controls. An initial report, suggesting that <it>HOXB1 </it>could confer autism vulnerability in interaction with <it>HOXA1</it>, was not confirmed by five small association studies.</p> <p>Methods</p> <p>Our sample includes 269 autistic individuals, belonging to 219 simplex and 28 multiplex families. A mutational analysis of the two exons and flanking intronic sequences of the <it>HOXB1 </it>gene was carried out in 84 autistic patients by denaturing high performance liquid chromatography, followed by DNA sequencing. Identified rare variants were then searched by a restriction analysis in 236 autistic patients and 325-345 controls. Case-control and family-based association studies were performed on two common variants in 169 Italian patients versus 184 Italian controls and in 247 trios.</p> <p>Results</p> <p>We identified three common polymorphisms, rs72338773 [c.82insACAGCGCCC (INS/nINS)], rs12939811 [c.309A>T (Q103H)], and rs7207109 [c.450G>A (A150A)] and three rare variants, namely IVS1+63G>A, rs35115415 [c.702G>A (V234V)] and c.872_873delinsAA (S291N). SNPs rs72338773 and rs12939811 were not associated with autism, using either a case-control (alleles, exact <it>P </it>= 0.13) or a family-based design [transmission/disequilibrium test (TDT)χ²= 1.774, <it>P </it>= 0.183]. The rare variants, all inherited from one of the parents, were present in two Italian and in two Caucasian-American families. Autistic probands in two families surprisingly inherited a distinct rare variant from each parent. The IVS1+63A allele was present in 3/690 control chromosomes, whereas rare alleles at rs35115415 and c.872_873delinsAA (S291N) were not found in 662 and 650 control chromosomes, respectively. The INS-T309 allele influenced head size, but its effect appears more modest and shows no interaction with <it>HOXA1 </it>alleles. The INS-T309 allele is also associated with more severe stereotypic behaviours, according to ADI-R scores (<it>N </it>= 60 patients, <it>P </it>< 0.01).</p> <p>Conclusions</p> <p><it>HOXB1 </it>mutations do not represent a common cause of autism, nor do <it>HOXB1 </it>common variants play important roles in autism vulnerability. <it>HOXB1 </it>provides minor, albeit detectable contributions to head circumference in autistic patients, with <it>HOXA1 </it>displaying more prominent effects. <it>HOXB1 </it>variants may modulate the clinical phenotype, especially in the area of stereotypic behaviours.</p

Relative HERV-H, W and K expression in ASD patients and healthy controls.

<p>Data obtained in fresh PBMCs (T₀) and after <i>in vitro</i> stimulation (T₇₂) of ASD patients and healthy controls are represented as box plot. ASD patients show higher levels of HERV-H (panel A) both at T₀ and after <i>in vitro</i> stimulation in comparison to healthy controls. Conversely, HERV-W (panel B) expression is higher in healthy controls than in ASD patients, both at T₀ and after <i>in vitro</i> stimulation. Significant differences between groups are shown. HERV-K (panel C) expression levels were similar in ASDs compared to HCs in both conditions, and significant differences were achieved only inside control group. Relative env gene expression levels were analyzed by Real-time PCR and represented by 2^−ΔΔCt in logarithmic scale.</p

Demographic information for the ASD and control cohorts.

<p>Demographic information for the ASD and control cohorts.</p

Clinical features and assessments obtained using the ADI-R, ADOS, CARS and PEP-3 diagnostic scales.

̂<p>ne: not evaluated.</p>*<p>ASS: Autism Severity Score.</p>1<p>: Communication;</p>2<p>: Motor;</p>3<p>: Maladaptative behaviors.</p

Quantitative evaluation of HERV-H expression in individual ASD patients and healthy controls.

<p>Comparative analysis of expression levels in individual ASD patients (panel A) shows that <i>in vitro</i> stimulation of PBMCs induces HERV-H expression increase in the patients with low levels at T₀. Conversely, <i>in vitro</i> stimulation does not modify HERV-H levels in individual HC (panel B). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048831#s3" target="_blank">Results</a> obtained by Real time PCR are represented as 2^−ΔΔCT in logarithmic scale. Red dashed line identifies values 2^−ΔΔCT>10.</p

Percentage of positive samples for expression of HERV families in cultured PBMCs from ASD and HC groups.

a<p>RT-PCR analysis of PBMCs at time 0 after withdrawal.</p>b<p>RT-PCR analysis of PBMCs after 72 hours of stimulation in culture.</p>c<p>RT-PCR analysis of PBMCs at T₀ and T₇₂.</p>d<p>No statistics are computed.</p><p>In parentheses the number of positive samples over the total (28).</p

Quantitative evaluation of HERV-W expression in individual ASD patients and healthy controls.

<p>No significant differences in expression levels of individual ASD patients (panel A) and healthy control (panel B) after <i>in vitro</i> stimulation are observed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048831#s3" target="_blank">Results</a> obtained by Real time PCR are represented as 2^−ΔΔCT>10 in logarithmic scale. Red dashed line identifies values 2^−ΔΔCT>10.</p

HERVs expression in autism spectrum disorders

Autistic Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental disorder, resulting from complex interactions among genetic, genomic and environmental factors. Here we have studied the expression of Human Endogenous Retroviruses (HERVs), non-coding DNA elements with potential regulatory functions, and have tested their possible implication in autism

Family-based association study of ITGB3 in autism spectrum disorder and its endophenotypes

The integrin-β 3 gene (ITGB3), located on human chromosome 17q21.3, was previously identified as a quantitative trait locus (QTL) for 5-HT blood levels and has been implicated as a candidate gene for autism spectrum disorder (ASD). We performed a family-based association study in 281 simplex and 12 multiplex Caucasian families. ITGB3 haplotypes are significantly associated with autism (HBAT, global P=0.038). Haplotype H3 is largely over-transmitted to the affected offspring and doubles the risk of an ASD diagnosis (HBAT P=0.005; odds ratio (OR)=2.000), at the expense of haplotype H1, which is under-transmitted (HBAT P=0.018; OR=0.725). These two common haplotypes differ only at rs12603582 located in intron 11, which reaches a P-value of 0.072 in single-marker FBAT analyses. Interestingly, rs12603582 is strongly associated with pre-term delivery in our ASD patients (P=0.008). On the other hand, it is SNP rs2317385, located at the 5′ end of the gene, that significantly affects 5-HT blood levels (Mann–Whitney U-test, P=0.001; multiple regression analysis, P=0.010). No gene–gene interaction between ITGB3 and SLC6A4 has been detected. In conclusion, we identify a significant association between a common ITGB3 haplotype and ASD. Distinct markers, located toward the 5′ and 3′ ends of the gene, seemingly modulate 5-HT blood levels and autism liability, respectively. Our results also raise interest into ITGB3 influences on feto–maternal immune interactions in autism