Whole-exome sequencing identifies genes associated with Tourette’s disorder in multiplex families

Tourette’s Disorder (TD) is a neurodevelopmental disorder (NDD) that affects about 0.7% of the population and is one of the most heritable NDDs. Nevertheless, because of its polygenic nature and genetic heterogeneity, the genetic etiology of TD is not well understood. In this study, we combined the segregation information in 13 TD multiplex families with high-throughput sequencing and genotyping to identify genes associated with TD. Using whole-exome sequencing and genotyping array data, we identified both small and large genetic variants within the individuals. We then combined multiple types of evidence to prioritize candidate genes for TD, including variant segregation pattern, variant function prediction, candidate gene expression, protein–protein interaction network, candidate genes from previous studies, etc. From the 13 families, 71 strong candidate genes were identified, including both known genes for NDDs and novel genes, such as HtrA Serine Peptidase 3 (HTRA3), Cadherin-Related Family Member 1 (CDHR1), and Zinc Finger DHHC-Type Palmitoyltransferase 17 (ZDHHC17). The candidate genes are enriched in several Gene Ontology categories, such as dynein complex and synaptic membrane. Candidate genes and pathways identified in this study provide biological insight into TD etiology and potential targets for future studies.This study was supported by a grant from the National Institute of Mental Health (R01MH092293 to GAH and JAT) and by a grant from the New Jersey Center for Tourette Syndrome (to GAH and JAT). This study was also supported by grants from the National Institute of Mental Health (K08MH099424 to TVF) and the National Institute for Environmental Health Science (R01 ES021462 for YSK and BLL). PM has received grants from the Instituto de Salud Carlos III (PI10/01674, PI13/01461), the Consejería de Economía, Innovación, Ciencia y Empresa de la Junta de Andalucía (CVI-02526, CTS-7685), the Consejería de Salud y Bienestar Social de la Junta de Andalucía (PI-0741/2010, PI-0437-2012, PI-0471-2013), the Sociedad Andaluza de Neurología, the Fundación Alicia Koplowitz, the Fundación Mutua Madrileña, and the Jaques and Gloria Gossweiler Foundation. AM has received grants from the Fundacion Alicia Koplowitz and belongs to the research group of the Comissionat per Universitats i Recerca del Departmanent d’Innovacio (DIUE) 2009SGR1119. AM has received grants from the Deutsche Forschungsgemeinschaft (DFG: MU 1692/3-1, MU 1692/4-1, and FOR 2698). AJW received a Young Investigator Award from Tourette Association of America. IH declares that all research at Great Ormond Street Hospital NHS Foundation Trust and UCL Great Ormond Street Institute of Child Health is made possible by the NIHR Great Ormond Street Hospital Biomedical Research Centre

De Novo Sequence and Copy Number Variants Are Strongly Associated with Tourette Disorder and Implicate Cell Polarity in Pathogenesis

We previously established the contribution of de novo damaging sequence variants to Tourette disorder (TD) through whole-exome sequencing of 511 trios. Here, we sequence an additional 291 TD trios and analyze the combined set of 802 trios. We observe an overrepresentation of de novo damaging variants in simplex, but not multiplex, families; we identify a high-confidence TD risk gene, CELSR3 (cadherin EGF LAG seven-pass G-type receptor 3); we find that the genes mutated in TD patients are enriched for those related to cell polarity, suggesting a common pathway underlying pathobiology; and we confirm a statistically significant excess of de novo copy number variants in TD. Finally, we identify significant overlap of de novo sequence variants between TD and obsessive-compulsive disorder and de novo copy number variants between TD and autism spectrum disorder, consistent with shared genetic risk

Effect of fetal alcohol exposure on D2R promoter methylation in the pituitary.

<p>A. Schematic representation of rat D2R promoter CpG island. Rat D2R promoter sequence was analyzed to search for CpG islands by Methyl primer v1 program. Each small vertical line represents single CpG. The thick solid bar below represents a CpG island. Arrows represent primer annealing sites (A). D2R promoter methylation in pituitaries of AD, PF, AF rats after ovariectomized and estrogen treatment for 60 or 90 days was performed by real time methylation specific PCR. Methylation was measured as ratio of methylated verses unmethylated DNA. D2R methylation in pituitary of AD, PF, AF rats after ovariectomized and estrogen treatment for 60 (B) and 90 days (C). Pyrosequencing analysis of 3 individual CpGs in the CpG island of the D2R promoter in pituitaries of AD, PF, AF rats after ovariectomized and estrogen treated for 60 days (D). Data are mean ± SEM of n = 6 animals per each group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. **, P<0.01 between AF and control AD or PF.</p

Fetal Alcohol Exposure Reduces Dopamine Receptor D2 and Increases Pituitary Weight and Prolactin Production via Epigenetic Mechanisms

<div><p>Recent evidence indicated that alcohol exposure during the fetal period increases the susceptibility to tumor development in mammary and prostate tissues. Whether fetal alcohol exposure increases the susceptibility to prolactin-producing tumor (prolactinoma) development in the pituitary was studied by employing the animal model of estradiol-induced prolactinomas in Fischer 344 female rats. We employed an animal model of fetal alcohol exposure that simulates binge alcohol drinking during the first two trimesters of human pregnancy and involves feeding pregnant rats with a liquid diet containing 6.7% alcohol during gestational day 7 to day 21. Control rats were pair-fed with isocaloric liquid diet or fed <i>ad libitum</i> with rat chow diet. Adult alcohol exposed and control female offspring rats were used in this study on the day of estrus or after estrogen treatment. Results show that fetal alcohol-exposed rats had increased levels of pituitary weight, pituitary prolactin (PRL) protein and mRNA, and plasma PRL. However, these rats show decreased pituitary levels of dopamine D2 receptor (D2R) mRNA and protein and increased pituitary levels of D2R promoter methylation. Also, they show elevated pituitary mRNA levels of DNA methylating genes (DNMT1, DNMT3b, MeCP2) and histone modifying genes (HDAC2, HDAC4, G9a). When fetal alcohol exposed rats were treated neonatally with a DNA methylation inhibitor 5-Aza deoxycytidine and/or a HDAC inhibitor trichostatin-A their pituitary D2R mRNA, pituitary weights and plasma PRL levels were normalized. These data suggest that fetal alcohol exposure programs the pituitary to increase the susceptibility to the development of prolactinomas possibly by enhancing the methylation of the D2R gene promoter and repressing the synthesis and control of D2R on PRL-producing cells.</p></div

Effects of fetal alcohol exposure on pituitary weight, plasma PRL and percentage of mitotic lactotropes in pituitaries of cyclic, ovariectomized and estrogen-treated ovariectomized animals.

<p>Alcohol-fed (AF), pair-fed (PF) or <i>ad libitum</i>-fed (AD) rat offsprings were used during the adult period (75–80 days) either on the day of estrus (cyclic) or 15 days after ovariectomy and empty implants (OVEX) or β-estradiol implants (E2-15 d). Mean ± SEM values of pituitary weight (A) and plasma PRL (B) in cyclic animals, pituitary weight (C) and plasma PRL (D) in OVEX or E2-15 d animals, and the percentage of mitotic lactotropes in pituitary of cyclic (E) and E2-15 d (F) animals are shown in the histograms. Data are mean ± SEM of n = 6–8 animals per each group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. **, P<0.01 and ***, P<0.001 between AF and controls (AD or PF).</p

Effects of epigenetic modulatory drugs on fetal alcohol induced changes in pituitary D2R mRNA levels, protein levels, pituitary weight and plasma PRL levels.

<p>AD, PF and AF rat offspring were treated with DNA methyl transferase inhibitor 5-Aza deoxycytidine (5-AZAdC), HDACC inhibitor Trichostatin-A (TSA) alone or together during postnatal day 2–6 and after 60 days, these rats were ovariectomized and estrogen treated for 60 days. Mean ± SEM values of pituitary D2R mRNA levels (A), pituitary weight (B) and plasma PRL levels (C) are shown in the histograms. Data are mean ± SEM of n = 6 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05, between AF and controls (AD or PF). @, P<0.05 between the treatment and control in AF group.</p

Schematic diagram illustrating fetal alcohol exposure induced epigenetic changes regulating D2R expression and its control of PRL synthesis and lactotropic cell growth in the pituitary.

<p>In control rats, D2R mRNA expression is regulated by the epigenetic mechanism involving DNA demethylation and histone deacetylation of the D2R promoter. D2R participates in mediating the inhibitory action of dopamine on PRL synthesis and cell proliferation in lactotropic cells of the pituitary gland (A). In fetal alcohol exposed (FAE) rats, increased D2R promoter methylation and histone deacetylation result in reduced D2R expression, The lower number of D2Rs prevents dopamine to act on lactotropes causing more PRL production and increased cell proliferation (B). DNA wrapped with histone octomer is represented as black thread with cylindrical structures. Methylated CpG is represented as pentagon structure in the DNA. Acetyl groups of histones are represented as triangles and methyl groups as pentagons on N terminal tails of histone.</p

Effect of fetal alcohol exposure on the sensitivity of pituitary lactotropes to the estrogen tumor-promoting action.

<p>Alcohol-fed (AF) or control-fed (PF, AD) rats offspring were ovariectomized and implanted with a β-estradiol implants at 60 days of age and after 60 (E2-60 d) or 90 days (E2-90 d) were used for this study. Mean ± SEM values of pituitary weight at 60 d (A) and 90 d (B), pituitary PRL mRNA level at 60 d (C) and 90 d (D), pituitary PRL protein level at 60 d (E) and 90 d (F), and plasma PRL levels at 60 d (G) and 90 d (H) are shown in the histograms. Data are mean ± SEM of n = 4–8 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05 and ***, P<0.001 between AF and controls (AD or PF).</p

Effect of fetal alcohol exposure on the level of genes regulating DNA methylation and histone modification in the pituitary.

<p>Pituitary mRNA levels of DNMT1 (A), DNMT3a (B), DNMT3b (C), MeCP2 (D), HDAC2 (E), HDAC4 (F), G91 (G) and Set7 (H) of AD, PF. AF rats after ovariectomy and estrogen treatment for 60 days. Data are mean ± SEM of n = 6 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05, **, P<0.01, and ***, P<0.001 between AF and controls (AD or PF).</p

Effect of fetal alcohol exposure on DNA methylation and histone deacetylation proteins in the pituitary.

<p>Pituitary protein levels of DNMT1 (A), DNMT3b (B), HDAC2 (C), HDAC4 (D) of AD, PF and AF rats after ovariectomy and estrogen treatment for 60 days. Data are mean + SEM of n = 6 animals per group and were analyzed using one-way ANOVA with the Newman-Keuls post hoc test. *, P<0.05, **, P<0.01 between AF and controls (AD or PF).</p