Somatic Overgrowth Predisposes to Seizures in Autism Spectrum Disorders

<div><p>Background</p><p>Comorbidity of Autism Spectrum Disorders with seizures or abnormal EEG (Autism-Epilepsy Phenotype) suggests shared pathomechanisms, and might be a starting point to identify distinct populations within the clinical complexity of the autistic spectrum. In this study, we tried to assess whether distinct subgroups, having distinctive clinical hallmarks, emerge from this comorbid condition.</p><p>Methods</p><p>Two-hundred and six individuals with idiopathic Autism Spectrum Disorders were subgrouped into three experimental classes depending on the presence of seizures and EEG abnormalities. Neurobehavioral, electroclinical and auxological parameters were investigated to identify differences among groups and features which increase the risk of seizures. Our statistical analyses used ANOVA, post-hoc multiple comparisons, and the Chi-squared test to analyze continuous and categorical variables. A correspondence analysis was also used to decompose significant Chi-squared and reduce variables dimensions.</p><p>Results</p><p>The high percentage of children with seizures (28.2% of our whole cohort) and EEG abnormalities (64.1%) confirmed that the prevalence of epilepsy in Autism Spectrum Disorders exceeds that of the general population. Seizures were associated with severe intellectual disability, and not with autism severity. Interestingly, tall stature (without macrocephaly) was significantly associated with EEG abnormalities or later onset seizures. However, isolated macrocephaly was equally distributed among groups or associated with early onset seizures when accompanied by tall stature.</p><p>Conclusions</p><p>Tall stature seems to be a phenotypic “biomarker” of susceptibility to EEG abnormalities or late epilepsy in Autism Spectrum Disorders and, when concurring with macrocephaly, predisposes to early onset seizures. Growth pattern might act as an endophenotypic marker in Autism-Epilepsy comorbidity, delineating distinct pathophysiological subtypes and addressing personalized diagnostic work-up and therapeutic approaches.</p></div

Distribution of Z-scores for height (Zh).

<p><b>A.</b> The histogram of Zh of whole sample shows a significant rightward shift (dashed curve) compared to the normative distribution (closed curve). <b>B.</b> The Zh distribution of ASD “simplex” group shows no difference with respect to normative distribution. <b>C.</b> The Zh distribution of ASD-EEG group shows a significant rightward shift (dashed curve) compared to the normative distribution (closed curve). <b>D.</b> The Zh distribution of ASD-seizures group shows a trend towards a rightward shift (dashed curve) compared to the normative distribution (closed curve). Arrows in C and D indicate a clearly higher rate of tall individuals over 1.5 SDS.</p

Zebrafish <i>stxbp1a</i> CRISPR/Cas9 mutant allele.

<p>(A) Sites of human and zebrafish mutations in highly conserved Stxbp1 sequence. Human STXBP1A protein sequence was aligned to zebrafish Stxbp1a and Stxbp1b. Black background indicates amino acid residues that are similar in all three proteins (BLOSUM 62). Grey background indicates amino acid residues that are similar between two of the three proteins. The salmon-colored background indicates the position of the deletion mutation in zebrafish mutant alleles. (B) Alignment of the mutant <i>stxbp1a</i>^s3000 allele sequence (top) with wild-type zebrafish <i>stxbp1a</i> sequence (bottom). The CRISPR/Cas9 target site is shown as a wide purple arrow below the plot. The site of the 4bp deletion <i>stxbp1a</i>^s3000 allele is highlighted in salmon, and corresponds to amino acids 211–212 highlighted in (A). (C) Alignment of the mutant <i>stxbp1b</i>^<i>s3001</i> allele sequence with wild-type zebrafish <i>stxbp1b</i> sequence.</p

Ontogeny of <i>stxbp1a</i> and <i>stxbp1b</i> expression in zebrafish larvae.

<p>Wild-type zebrafish were probed for expression of <i>stxbp1a</i> mRNA (A-E) or <i>stxbp1b</i> mRNA (F-J). Abbreviations: CeP: cerebellar plate, Ha: habenula, IR: inner retina, MO: medulla oblongata, OB: olfactory bulb, P: pallium, Ret: retina, SC: spinal cord, Tel: telencephalon, TeO: optic tectum, Scale bar = 100μm.</p

Metabolic and survival deficits caused by <i>stxbp1a</i>^s3000 mutation.

<p>Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae have lower metabolism than controls (siblings) and die prematurely. (A) 5 dpf <i>stxbp1a</i>^{<i>s3000/s3000</i>} homozygous mutants (n = 10) had significantly lower extracellular acidification (ECAR) than siblings (n = 10; mean ± SD). * = p < 0.0001 (two-tailed t-test). (B) 5 dpf <i>stxbp1a</i>^{<i>s3000/s3000</i>} homozygous mutants (n = 10) had significantly lower oxygen consumption (OCR) than siblings (n = 10; mean ± SD). * = p < 0.0001 (two-tailed t-test). (C) Heart rates of <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae (n = 15) at 3 dpf were significantly lower than heart rates of their siblings (n = 33). All measured values are plotted; mean values are indicated by horizontal lines. (D) Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutants die as larvae. Homozygous mutants (n = 50) and siblings (n = 50) were maintained in petri dishes without food and counted each day from 2 dpf until 10dpf. The <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutants began dying at 6 dpf, and 98% (49) died by 10dpf. Only 2% (1) of siblings died at 10dpf.</p

Auxological parameters.

<p><b>AUX1</b>: Normal head circumference and height; <b>AUX2</b>: Isolated Macrocephaly; <b>AUX3</b>: Macrocephaly and Tall Stature; <b>AUX4</b>: Isolated Tall Stature.</p>*<p>Tall Stature is strongly unrelated to the ASD “simplex” group, and is associated with the ASD-EEG group (see CA in Table S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075015#pone.0075015.s001" target="_blank">Supporting Information S1</a>). <b>**</b>Isolated Tall Stature (AUX4) is associated with EEG abnormalities; concurrence of Tall Stature and Macrocephaly (AUX3) is associated to seizures; Isolated Macrocephaly (AUX 2) is equally distributed among groups (see CA in Table S6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075015#pone.0075015.s001" target="_blank">Supporting Information S1</a>).</p><p><b>Φc = </b> Cramers’ phi coefficient, <b>χ2 = </b>the Pearson chi-squared test.</p

Characteristics of total sample and experimental groups.

*<p>one-way ANOVA, post-hoc Bonferroni method, ASD-seizures > ASD-EEG, p<0.001; ASD-seizures > ASD “simplex”, p<0.001; ASD-EEG vs ASD “simplex”, p>0.010; <b>**</b>EEG abnormalities are significantly associated with the presence of seizures (56/58 children with seizures vs 76/148 without seizures).</p><p><b>Φc = </b> Cramers’ phi coefficient, <b>η = </b>eta, <b>χ² = </b>the Pearson chi-squared test, <b>F = </b> F of Fisher, one-way ANOVA.</p

Normal metabolic function in <i>stxbp1b</i>^<i>s3001</i> mutants.

<p>Homozygous <i>stxbp1b</i>^{<i>s30010/s3001</i>} mutant larvae have unaffected metabolism compared to controls (siblings). (A) 5 dpf <i>stxbp1b</i>^{<i>s30010/s3001</i>} homozygous mutants (n = 11) did not differ from siblings (n = 52) in extracellular acidification (ECAR) (two-tailed t-test; mean ± SD). (B) 5 dpf <i>stxbp1b</i>^{<i>s30010/s3001</i>} homozygous mutants (n = 11) did not differ from siblings (n = 52) in oxygen consumption (OCR) (two-tailed t-test; mean ± SD). (C) Heart rates of <i>stxbp1b</i>^{<i>s30010/s3001</i>} mutant larvae (n = 16) at 3 dpf were not significantly different from heart rates of their siblings (n = 34). All measured values are plotted; mean values are indicated by horizontal lines. (D) All <i>stxbp1b</i>^{<i>s30010/s3001</i>} mutant larvae tested (n = 8) survived until 10 dpf; 12.5% of their control siblings (n = 48) died by 10dpf.</p

Biplot for Cross-Tabulation of Auxological Categories by Experimental Groups.

<p>Correspondence analysis at two-dimensional solution showed that ASD-EEG group is relatively associated with Isolated Tall Stature (AUX4, closed circle), and ASD-seizures group is relatively associated with combined macrocephaly and tall stature (AUX3, dashed circle).</p

Morphology of <i>stxbp1a</i>^s3000 mutant zebrafish.

<p>(A) Heterozygous <i>stxbp1a</i>^{<i>s3000/+</i>} mutant larvae (5 dpf) are morphologically indistinguishable from wild-type siblings. Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae are immobile and fail to hatch out of the chorion. Scale bar = 500 μm. (B) Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae (n = 10) removed from their chorions are not significantly different in length from their siblings (n = 30; p = 0.0592, two-tailed t-test). (C-D) At 5 dpf, the dorsal surface of homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae that were removed from their chorions at 2 dpf (D) show dispersed melanin and foreshortened craniofacial structure compared to siblings (C). Scale bar = 300 μm.</p