Increased Expression of the Dyslexia Candidate Gene DCDC2 Affects Length and Signaling of Primary Cilia in Neurons

Peer reviewe

The complex of TFII-I, PARP1, and SFPQ proteins regulates the DYX1C1 gene implicated in neuronal migration and dyslexia

DYX1C1 was first identified as a candidate gene for dyslexia susceptibility, and its role in controlling neuronal migration during embryogenesis and effect on learning in rodents have been verified. In contrast, genetic association studies have been ambiguous in replicating its effects on dyslexia. To better understand the regulation of DYX1C1 and the possible functional role of genetic variation in the promoter of DYX1C1, we selected three single-nucleotide polymorphisms (SNPs) with predicted functional consequences or suggested associations to dyslexia for detailed study. Electrophoretic mobility shift assays suggested the allele-specific binding of the transcription factors TFII-I (to rs3743205) and Sp1 (to rs16787 and rs12899331) that could be verified by competition assays. In addition, we purified a complex of protein factors binding to the previously suggested dyslexia-related SNP, −3G/A (rs3743205). Three proteins, TFII-I, PARP1, and SFPQ, were unambiguously identified by mass spectrometry and protein sequencing. Two SNPs, rs16787 and rs3743205, showed significant allelic differences in luciferase assays. Our results show that TFII-I, PARP1, and SFPQ proteins, each previously implicated in gene regulation, form a complex controlling transcription of DYX1C1. Furthermore, allelic differences in the promoter or 5′ untranslated region of DYX1C1 may affect factor binding and thus regulation of the gene.—Tapia-Páez, I., Tammimies, K., Massinen, S., Roy. A. L., Kere, J. The complex of TFII-I, PARP1, and SFPQ proteins regulates the DYX1C1 gene implicated in neuronal migration and dyslexia

DNA copy-number analysis of the 22q11 deletion-syndrome region using array-CGH with genomic and PCR-based targets

Deletions and duplications of genomic segments commonly cause developmental disorders. The resolution and efficiency in diagnosing such gene-dosage alterations can be drastically increased using microarray-based comparative genomic hybridization (array-CGH). However, array-CGH currently relies on spotting genomic clones as targets, which confers severe limitations to the approach including resolution of analysis and reliable gene-dosage assessment of regions with high content of redundant sequences. To improve the methodology for analysis, we compared the use of genomic clones, repeat-free pools of amplified genomic DNA and cDNAs (single and pooled) as targets on the array. For this purpose, we chose q11.2 locus on chromosome 22 as a testing ground. Microdeletions at 22q11 cause birth defects collectively described as the DiGeorge/velocardiofacial syndrome. The majority of patients present 3 Mb typical deletions. Here, we report the construction of a gene-dosage array, covering 6 Mb of 22q11 and including the typically deleted region. We hybridized DNA from six DiGeorge syndrome patients to the array, and show that as little as 11.5 kb non-redundant, repeat-free PCR-generated sequence can be used for reliable detection of hemizygous deletions. By extrapolation, this would allow analysis of the genome with an average resolution of 25 kb. In the case of cDNAs our results indicate that 3.5 kb sequence is necessary for accurate identification of haploid/diploid dosage alterations. Thus, for regions rich in redundant sequences and repeats, such as 22q11, a specifically tailored array-CGH approach is good for gene copy number profiling

Sequence alignment and genomic structure of the vertebrate <i>DYX1C1</i>/<i>Dyx1c1</i>/<i>dyx1c1</i> genes and proteins.

<p>Sequence comparison of zebrafish (<i>Danio rerio</i>; Dre) Dyx1c1 protein with that of Human (<i>Homo sapiens</i>; Hs) and mouse (<i>Mus musculus</i>; Ms) protein sequences (A). The p23, DYX and TPR domains are denoted by red, black and blue lines respectively. Protein sequence identity shown in percentage for the three species (B). The comparison of <i>dyx1c1</i> cDNAs from Human, mouse and zebrafish reveals 9 exons intercepted by 8 introns (C). Human cDNA sequence shows the presence of an additional intron in the 5′untranslated region (UTR). The UTRs are denoted by green boxes, exons by blue boxes with the number of amino acids they code and introns are represented by slanting lines connecting the exons with the number of nucleotides (nt) on top. The conserved domains are denoted by red (for p23 domain), black (for DYX domain) and blue (for TPR domain) lines below the cDNA structure for each species.</p

Knockdown of <i>dyx1c1</i> showed typical cilia phenotypes.

<p>Injection of ATGMO or SPMO at 200 µM concentration produced ventrally curved body axis, hydrocephalus and kidney cysts (A). Arrow in panel A denotes kidney cyst in ATG morphant. RT-PCR showed aberrant splice transcripts in SPMO injected embryos at 1 and 2 dpf (B). Histological sections of 2 day old embryos injected with both ATGMO and SPMO (100 µM each) showed hydrocephalus (D; yellow arrows) compared to normal size brain ventricles in WT (C). Transverse histological sections across the pronephros at 3.5 dpf showed normal pronephros in WT embryos (E). Section of <i>dyx1c1</i> morphant (ATGMO+SPMO) showed severe pronephric distention and a thin glomerulus in the center (F). Yellow arrowheads in panel E and F point out normal pronephros in WT and dilated pronephros in morphant embryo, respectively. Quantitative analysis of the rescue of <i>dyx1c1</i> morphant phenotype to WT phenotype with different combinations of MOs and <i>dyx1c1</i> mRNA (G). Scale bars indicate 100 µm. Abbreviation: gm, glomerulus.</p

Expression of <i>dyx1c1</i> mRNA during embryonic development and in adult tissues.

<p>qPCR analysis of the transcript levels of <i>dyx1c1</i> during embryonic development (A) and in adult tissues (B). Whole-mount <i>in situ</i> hybridization showed that <i>dyx1c1</i> is expressed in KV at 10 hpf (C). Inset in C is a close-up view of KV. At 15-somites, <i>dyx1c1</i> was expressed specifically in the otic vesicle, pronephros and neural tube (D). At 26 hpf <i>dyx1c1</i> was detected in the brain and is still maintained in otic vesicle, pronephros and spinal canal (E–G). Later at 49 hpf, <i>dyx1c1</i> was visible in the olfactory placode (H). Panels E–G show lateral views of embryos. Panels D and H show dorsal and ventral views of embryos, respectively. Scale bars indicate 100 µm. Abbreviations: KV, Kupffer’s vesicle: nt, neural tube: pn, pronephros: t, telencephalon: d, diencephalon: m, midbrain: tg, tegmentum: ov, otic vesicle: sc, spinal canal: op, olfactory placode.</p

DCDC2 mutations cause a renal-hepatic ciliopathy by disrupting Wnt signaling

Item does not contain fulltextNephronophthisis-related ciliopathies (NPHP-RC) are recessive diseases characterized by renal dysplasia or degeneration. We here identify mutations of DCDC2 as causing a renal-hepatic ciliopathy. DCDC2 localizes to the ciliary axoneme and to mitotic spindle fibers in a cell-cycle-dependent manner. Knockdown of Dcdc2 in IMCD3 cells disrupts ciliogenesis, which is rescued by wild-type (WT) human DCDC2, but not by constructs that reflect human mutations. We show that DCDC2 interacts with DVL and DCDC2 overexpression inhibits beta-catenin-dependent Wnt signaling in an effect additive to Wnt inhibitors. Mutations detected in human NPHP-RC lack these effects. A Wnt inhibitor likewise restores ciliogenesis in 3D IMCD3 cultures, emphasizing the importance of Wnt signaling for renal tubulogenesis. Knockdown of dcdc2 in zebrafish recapitulates NPHP-RC phenotypes, including renal cysts and hydrocephalus, which is rescued by a Wnt inhibitor and by WT, but not by mutant, DCDC2. We thus demonstrate a central role of Wnt signaling in the pathogenesis of NPHP-RC, suggesting an avenue for potential treatment of NPHP-RC