Anophthalmia/microphthalmia (A/M) is a genetically heterogeneous birth defect for which the etiology is unknown in more than 50% of patients. We used exome sequencing with the ACE Exome™ (Personalis, Inc; 18 cases) and UCSF Genomics Core (21 cases) to sequence 28 patients with A/M and four patients with varied developmental eye defects. In the 28 patients with A/M, we identified de novo mutations in three patients (OTX2, p.(Gln91His), RARB, p.Arg387Cys and GDF6, p.Ala249Glu) and inherited mutations in STRA6 in two patients. In patients with developmental eye defects, a female with cataracts and cardiomyopathy had a de novo COL4A1 mutation, p.(Gly773Arg), expanding the phenotype associated with COL4A1 to include cardiomyopathy. A male with a chorioretinal defect, microcephaly, seizures and sensorineural deafness had two PNPT1 mutations, p.(Ala507Ser) and c.401-1G>A, and we describe eye defects associated with this gene for the first time. Exome sequencing was efficient for identifying mutations in pathogenic genes for which there is no clinical testing available and for identifying cases that expand phenotypic spectra, such as the PNPT1 and COL4A1-associated disorders described here.
SUMMARY We demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2+/− sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex; ventral expansion of the dorsomedial cortex; ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-Seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate.
BackgroundCardiovascular disease (CVD) is the leading cause of death in the developed world. Human genetic studies, including genome-wide sequencing and SNP-array approaches, promise to reveal disease genes and mechanisms representing new therapeutic targets. In practice, however, identification of the actual genes contributing to disease pathogenesis has lagged behind identification of associated loci, thus limiting the clinical benefits.ResultsTo aid in localizing causal genes, we develop a machine learning approach, Objective Prioritization for Enhanced Novelty (OPEN), which quantitatively prioritizes gene-disease associations based on a diverse group of genomic features. This approach uses only unbiased predictive features and thus is not hampered by a preference towards previously well-characterized genes. We demonstrate success in identifying genetic determinants for CVD-related traits, including cholesterol levels, blood pressure, and conduction system and cardiomyopathy phenotypes. Using OPEN, we prioritize genes, including FLNC, for association with increased left ventricular diameter, which is a defining feature of a prevalent cardiovascular disorder, dilated cardiomyopathy or DCM. Using a zebrafish model, we experimentally validate FLNC and identify a novel FLNC splice-site mutation in a patient with severe DCM.ConclusionOur approach stands to assist interpretation of large-scale genetic studies without compromising their fundamentally unbiased nature.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0534-8) contains supplementary material, which is available to authorized users.
Cleft lip and/or palate (CL/P) are common structural birth defects in humans. We used exome sequencing to study a patient with bilateral CL/P and identified a single nucleotide deletion in the patient and her similarly affected son—c.546_546delG, predicting p.Gln183Argfs*57 in the Distal-less 4 (DLX4) gene. The sequence variant was absent from databases, predicted to be deleterious and was verified by Sanger sequencing. In mammals, there are three Dlx homeobox clusters with closely located gene pairs (Dlx1/Dlx2, Dlx3/Dlx4, Dlx5/Dlx6). In situ hybridization showed that Dlx4 was expressed in the mesenchyme of the murine palatal shelves at E12.5, prior to palate closure. Wild-type human DLX4, but not mutant DLX4_c.546delG, could activate two murine Dlx conserved regulatory elements, implying that the mutation caused haploinsufficiency. We showed that reduced DLX4 expression after short interfering RNA treatment in a human cell line resulted in significant up-regulation of DLX3, DLX5 and DLX6, with reduced expression of DLX2 and significant up-regulation of BMP4, although the increased BMP4 expression was demonstrated only in HeLa cells. We used antisense morpholino oligonucleotides to target the orthologous Danio rerio gene, dlx4b, and found reduced cranial size and abnormal cartilaginous elements. We sequenced DLX4 in 155 patients with non-syndromic CL/P and CP, but observed no sequence variants. From the published literature, Dlx1/Dlx2 double homozygous null mice and Dlx5 homozygous null mice both have clefts of the secondary palate. This first finding of a DLX4 mutation in a family with CL/P establishes DLX4 as a potential cause of human clefts.
Endothelin signaling is essential for neural crest development, and dysregulated Endothelin signaling is associated with several neural crest-related disorders, including Waardenburg and other syndromes. However, despite the crucial roles of this pathway in neural crest development and disease, the transcriptional effectors directly activated by Endothelin signaling during neural crest development remain incompletely elucidated. Here, we establish that the MADS box transcription factor MEF2C is an immediate downstream transcriptional target and effector of Endothelin signaling in the neural crest. We show that Endothelin signaling activates Mef2c expression in the neural crest through a conserved enhancer in the Mef2c locus and that CRISPR-mediated deletion of this Mef2c neural crest enhancer from the mouse genome abolishes Endothelin induction of Mef2c expression. Moreover, we demonstrate that Endothelin signaling activates neural crest expression of Mef2c by de-repressing MEF2C activity through a Calmodulin-CamKII-histone deacetylase signaling cascade. Thus, these findings identify a MEF2C-dependent, positive-feedback mechanism for Endothelin induction and establish MEF2C as an immediate transcriptional effector and target of Endothelin signaling in the neural crest.
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