CHD2 haploinsufficiency is associated with developmental delay, intellectual disability, epilepsy and neurobehavioural problems

BACKGROUND: The chromodomain helicase DNA binding domain (CHD) proteins modulate gene expression via their ability to remodel chromatin structure and influence histone acetylation. Recent studies have shown that CHD2 protein plays a critical role in embryonic development, tumor suppression and survival. Like other genes encoding members of the CHD family, pathogenic mutations in the CHD2 gene are expected to be implicated in human disease. In fact, there is emerging evidence suggesting that CHD2 might contribute to a broad spectrum of neurodevelopmental disorders. Despite growing evidence, a description of the full phenotypic spectrum of this condition is lacking. METHODS: We conducted a multicentre study to identify and characterise the clinical features associated with haploinsufficiency of CHD2. Patients with deletions of this gene were identified from among broadly ascertained clinical cohorts undergoing genomic microarray analysis for developmental delay, congenital anomalies and/or autism spectrum disorder. RESULTS: Detailed clinical assessments by clinical geneticists showed recurrent clinical symptoms, including developmental delay, intellectual disability, epilepsy, behavioural problems and autism-like features without characteristic facial gestalt or brain malformations observed on magnetic resonance imaging scans. Parental analysis showed that the deletions affecting CHD2 were de novo in all four patients, and analysis of high-resolution microarray data derived from 26,826 unaffected controls showed no deletions of this gene. CONCLUSIONS: The results of this study, in addition to our review of the literature, support a causative role of CHD2 haploinsufficiency in developmental delay, intellectual disability, epilepsy and behavioural problems, with phenotypic variability between individuals

Rare exonic deletions implicate the synaptic organizer Gephyrin (GPHN) in risk for autism, schizophrenia and seizures

The GPHN gene codes for gephyrin, a key scaffolding protein in the neuronal postsynaptic membrane, responsible for the clustering and localization of glycine and GABA receptors at inhibitory synapses. Gephyrin has well-established functional links with several synaptic proteins that have been implicated in genetic risk for neurodevelopmental disorders such as autism spectrum disorder (ASD), schizophrenia and epilepsy including the neuroligins (NLGN2, NLGN4), the neurexins (NRXN1, NRXN2, NRXN3) and collybistin (ARHGEF9). Moreover, temporal lobe epilepsy has been linked to abnormally spliced GPHN mRNA lacking exons encoding the G-domain of the gephyrin protein, potentially arising due to cellular stress associated with epileptogenesis such as temperature and alkalosis. Here, we present clinical and genomic characterization of six unrelated subjects, with a range of neurodevelopmental diagnoses including ASD, schizophrenia or seizures, who possess rare de novo or inherited hemizygous microdeletions overlapping exons of GPHN at chromosome 14q23.3. The region of common overlap across the deletions encompasses exons 3-5, corresponding to the G-domain of the gephyrin protein. These findings, together with previous reports of homozygous GPHN mutations in connection with autosomal recessive molybdenum cofactor deficiency, will aid in clinical genetic interpretation of the GPHN mutation spectrum. Our data also add to the accumulating evidence implicating neuronal synaptic gene products as key molecular factors underlying the etiologies of a diverse range of neurodevelopmental conditions

Ectopic <i>TLX1</i> Expression Accelerates Malignancies in Mice Deficient in <i>DNA-PK</i>

<div><p>The noncluster homeobox gene <i>HOX11/TLX1</i> (<i>TLX1</i>) is detected at the breakpoint of the t(10;14)(q24;q11) chromosome translocation in patients with T cell acute lymphoblastic leukemia (T-ALL). This translocation results in the inappropriate expression of <i>TLX1</i> in T cells. The oncogenic potential of <i>TLX1</i> was demonstrated in <i>IgHμ-TLX1^Tg</i> mice which develop mature B cell lymphoma after a long latency period, suggesting the requirement of additional mutations to initiate malignancy. To determine whether dysregulation of genes involved in the DNA damage response contributed to tumor progression, we crossed <i>IgHμ-TLX1^Tg</i> mice with mice deficient in the DNA repair enzyme DNA-PK (<i>Prkdc^Scid/Scid</i> mice). <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice developed T-ALL and acute myeloid leukemia (AML) with reduced latency relative to control <i>Prkdc^Scid/Scid</i> mice. Further analysis of thymi from premalignant mice revealed greater thymic cellularity concomitant with increased thymocyte proliferation and decreased apoptotic index. Moreover, premalignant and malignant thymocytes exhibited impaired spindle checkpoint function, in association with aneuploid karyotypes. Gene expression profiling of premalignant <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> thymocytes revealed dysregulated expression of cell cycle, apoptotic and mitotic spindle checkpoint genes in double negative 2 (DN2) and DN3 stage thymocytes. Collectively, these findings reveal a novel synergy between TLX1 and impaired DNA repair pathway in leukemogenesis.</p></div

Heat map of the top ranking differentially expressed genes in flow sorted premalignant thymocytes.

<p>(A) Venn diagrams depicting TLX1-associated up-regulated and down-regulated genes in DN1, DN2 and DN3 fractions. (B–D) Gene expression heat map of the top ranking differentially expressed genes in DN1 (B), DN2 (C) and DN3 (D) flow-sorted thymocytes from <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> mice. For each heat map, the first four columns (<i>TLX1</i>⁺) show <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> thymocyte-derived samples, and the last four columns (<i>TLX1</i>^-) represent samples derived from thymocytes of <i>Prkdc^Scid/Scid</i> mice.</p

Expression of <i>TLX1</i> in <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> premalignant thymocytes increases cell viability and provides a proliferative advantage.

<p>(A) Absolute cell numbers of thymocytes isolated from 20, six week old <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and 20 <i>Prkdc^Scid/Scid</i> littermates (p<0.0001). (B) Thymocytes were flow sorted based on expression of CD44 and CD25 and absolute numbers of DN thymocytes were calculated using percentages obtained after flow sorting (p<0.0001). (C) Thymocytes from three, six week old <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> littermates were stained with Annexin V and PI than assessed by flow cytometric analysis for cell viability. The lower left quadrant of each panel contains viable cells, the upper right quadrant contains dead cells and the lower right quadrant contains early apoptotic cells. The percentage of cells in each quadrant is indicated. (D) Percentages of viable, apoptotic and dead thymocytes in thymi of <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> littermates, as determined by flow cytometry with Annexin V and PI staining. Error bars represent SD. (E) Histogram showing premalignant thymocytes obtained from <i>Prkdc^Scid/Scid</i> and <i>IgHµ</i>-<i>TLX1^TgPrkdc^Scid/Scid</i> mice, 2 and 7 hours after intraperitoneal injection with 10 µM BrdU. (F) The percentages of proliferating cells in thymi of <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> and <i>Prkdc^Scid/Scid</i> littermates as determined by BrdU and PI staining. Data represent means of triplicate measurements with error bars to represent ± SD (p<0.0001). Statistically significant differences between compared samples are indicated by asterisks.</p

Chromosome analysis of premalignant thymocytes and tumors from <i>Prkdc^Scid/Scid</i> and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice.

<p>(A) Representative micrographs of Giemsa stained diploid, hyperdiploid, and hypoploid chromosome spreads from <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice. (B) Ploidy assessment of cultured thymocytes and thymic tumors obtained from premalignant and moribund <i>Prkdc^Scid/Scid</i> and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice, respectively. For each of three samples, 40 to 100 metaphase spreads were analyzed. The percentage of aneuploid spreads relative to the total number of analyzed spreads was determined. Statistically significant differences (p<0.05) are indicated by asterisks. (C) Spectral karyotype analysis of a hypoploid tumor isolated from an <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mouse with T-ALL. Loss of chromosome 12 and gain of chromosome 17 were found in 10% and 5% of analyzed cells, respectively. The karyotype is indicated below. (D) An abnormal, unbalanced trisomy 15 karyotype with a rob(5;15) translocation and two normal copies of chromosome 15. Examples of the rob(5;15) chromosomes and chromosome 15 are shown. The karyotype is indicated below.</p

qRT-PCR analysis of thymic tumors derived from <i>Prkdc^Scid/Scid</i> and <i>IgHμ-TLX1^TgPrkdc^Scid/Scid</i> mice.

<p>qRT-PCR analysis of expression of <i>Bcl11b</i>, <i>Pten</i> and <i>Notch1</i> in tumors isolated from ten <i>Prkdc^Scid/Scid</i> and ten <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice. Data were normalized relative to <i>β-actin</i>. Each bar represents one tumor sample.</p

Aberrant checkpoint regulation in thymocytes of <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice.

<p>(A) Cell cycle analysis showing the percentage of thymocytes in S and G2/M as determined by PI staining for DNA content. Percentages of cells in S and G2/M are shown above the histograms. (B) BrdU labeling to assess bypass of the G2/M cell cycle checkpoint in <i>Prkdc^Scid/Scid</i> and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> thymocyte cultures. Thymocytes were treated with colchicine to induce mitotic arrest then exposed to BrdU to assess cell cycling. BrdU incorporation was detected by BrdU immunolabeling and nulcei were revealed by DAPI staining. White arrows on the merged images indicate cycling thymocytes. The histogram depicts the mean percentages of BrdU-positive cells assessed by scoring 20 random fields. Statistically significant differences (p<0.05) are indicated by asterisks.</p

Immunophenotype of <i>TLX1</i>-induced T-ALL.

<p>Immunophenotype distribution showing heterogeneous expression of CD44, CD25, CD4 and CD8 in <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> T-ALL. Representative flow diagrams showing heterogeneous expression of CD4 and CD8 in <i>TLX1</i>-initiated leukemia are presented.</p

<i>TLX1-</i>induced T-ALL in <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice.

<p>(A) Hematoxylin and eosin staining of tissues isolated from premalignant <i>Prkdc^Scid/Scid</i> and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice and <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mice diagnosed with T-ALL. Magnification x40 (overview) and x100 (insert). Scale bars, 10 µm. (B) Immunohistochemical analysis of thymus and spleen from a premalignant <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mouse and a moribund <i>IgHµ-TLX1^TgPrkdc^Scid/Scid</i> mouse stained with an anti-Thy1.2 antibody. Magnification x20. Scale bars, 10 µm. (C) Cells from thymi, spleens and bone marrow of premalignant and moribund <i>IgHµ</i>-<i>TLX1^TgPrkdc^Scid/Scid</i> mice were examined for cell surface expression of CD44, CD25, CD4, CD8, CD3, TCRαβ, TCRγδ and Thy1.2 (for T cells) and Gr-1 and Mac-1 (for myeloid cells).</p