Array genotyping as diagnostic approach in medical genetics.

Genotyping arrays are by far the most widely used genetic tests but are not generally utilized for diagnostic purposes in a medical context. In the present study, we examined the diagnostic value of a standard genotyping array (Illumina Global Screening Array) for a range of indications. Applications included stand-alone testing for specific variants (32 variants in 10 genes), first-tier array variant screening for monogenic conditions (10 different autosomal recessive metabolic diseases), and diagnostic workup for specific conditions caused by variants in multiple genes (suspected familial breast and ovarian cancer, and hypercholesterolemia). Our analyses showed a high analytical sensitivity and specificity of array-based analyses for validated and non-validated variants, and identified pitfalls that require attention. Ethical-legal assessment highlighted the need for a software solution that allows for individual indication-based consent and the reliable exclusion of non-consented results. Cost/time assessment revealed excellent performance of diagnostic array analyses, depending on indication, proband data, and array design. We have implemented some analyses in our diagnostic portfolio, but array optimization is required for the implementation of other indications.

Spatially resolved RNA-sequencing of the embryonic heart identifies a role for Wnt/ß-catenin signaling in autonomic control of heart rate.

Development of specialized cells and structures in the heart is regulated by spatially -restricted molecular pathways. Disruptions in these pathways can cause severe congenital cardiac malformations or functional defects. To better understand these pathways and how they regulate cardiac development we used tomo-seq, combining high-throughput RNA-sequencing with tissue-sectioning, to establish a genome-wide expression dataset with high spatial resolution for the developing zebrafish heart. Analysis of the dataset revealed over 1100 genes differentially expressed in sub-compartments. Pacemaker cells in the sinoatrial region induce heart contractions, but little is known about the mechanisms underlying their development. Using our transcriptome map, we identified spatially restricted Wnt/ß-catenin signaling activity in pacemaker cells, which was controlled by Islet-1 activity. Moreover, Wnt/ß-catenin signaling controls heart rate by regulating pacemaker cellular response to parasympathetic stimuli. Thus, this high-resolution transcriptome map incorporating all cell types in the embryonic heart can expose spatially restricted molecular pathways critical for specific cardiac functions.

On the Evolution of the Cardiac Pacemaker.

The rhythmic contraction of the heart is initiated and controlled by an intrinsic pacemaker system. Cardiac contractions commence at very early embryonic stages and coordination remains crucial for survival. The underlying molecular mechanisms of pacemaker cell development and function are still not fully understood. Heart form and function show high evolutionary conservation. Even in simple contractile cardiac tubes in primitive invertebrates, cardiac function is controlled by intrinsic, autonomous pacemaker cells. Understanding the evolutionary origin and development of cardiac pacemaker cells will help us outline the important pathways and factors involved. Key patterning factors, such as the homeodomain transcription factors Nkx2.5 and Shox2, and the LIM-homeodomain transcription factor Islet-1, components of the T-box (Tbx), and bone morphogenic protein (Bmp) families are well conserved. Here we compare the dominant pacemaking systems in various organisms with respect to the underlying molecular regulation. Comparative analysis of the pathways involved in patterning the pacemaker domain in an evolutionary context might help us outline a common fundamental pacemaker cell gene programme. Special focus is given to pacemaker development in zebrafish, an extensively used model for vertebrate development. Finally, we conclude with a summary of highly conserved key factors in pacemaker cell development and function.

Mutations in SGOL1 cause a novel cohesinopathy affecting heart and gut rhythm.

The pacemaking activity of specialized tissues in the heart and gut results in lifelong rhythmic contractions. Here we describe a new syndrome characterized by Chronic Atrial and Intestinal Dysrhythmia, termed CAID syndrome, in 16 French Canadians and 1 Swede. We show that a single shared homozygous founder mutation in SGOL1, a component of the cohesin complex, causes CAID syndrome. Cultured dermal fibroblasts from affected individuals showed accelerated cell cycle progression, a higher rate of senescence and enhanced activation of TGF-ß signaling. Karyotypes showed the typical railroad appearance of a centromeric cohesion defect. Tissues derived from affected individuals displayed pathological changes in both the enteric nervous system and smooth muscle. Morpholino-induced knockdown of sgol1 in zebrafish recapitulated the abnormalities seen in humans with CAID syndrome. Our findings identify CAID syndrome as a novel generalized dysrhythmia, suggesting a new role for SGOL1 and the cohesin complex in mediating the integrity of human cardiac and gut rhythm.

Identification and functional characterization of cardiac pacemaker cells in zebrafish.

In the mammalian heart a conduction system of nodes and conducting cells generates and transduces the electrical signals evoking myocardial contractions. Specialized pacemaker cells initiating and controlling cardiac contraction rhythmicity are localized in an anatomically identifiable structure of myocardial origin, the sinus node. We previously showed that in mammalian embryos sinus node cells originate from cardiac progenitors expressing the transcription factors T-box transcription factor 3 (Tbx3) and Islet-1 (Isl1). Although cardiac development and function are strikingly conserved amongst animal classes, in lower vertebrates neither structural nor molecular distinguishable components of a conduction system have been identified, questioning its evolutionary origin. Here we show that zebrafish embryos lacking the LIM/homeodomain-containing transcription factor Isl1 display heart rate defects related to pacemaker dysfunction. Moreover, 3D reconstructions of gene expression patterns in the embryonic and adult zebrafish heart led us to uncover a previously unidentified, Isl1-positive and Tbx2b-positive region in the myocardium at the junction of the sinus venosus and atrium. Through their long interconnecting cellular protrusions the identified Isl1-positive cells form a ring-shaped structure. In vivo labeling of the Isl1-positive cells by transgenic technology allowed their isolation and electrophysiological characterization, revealing their unique pacemaker activity. In conclusion we demonstrate that Isl1-expressing cells, organized as a ring-shaped structure around the venous pole, hold the pacemaker function in the adult zebrafish heart. We have thereby identified an evolutionary conserved, structural and molecular distinguishable component of the cardiac conduction system in a lower vertebrate.

MicroRNA-23 restricts cardiac valve formation by inhibiting Has2 and extracellular hyaluronic acid production.

RATIONALE: Since their discovery almost 20 years ago, microRNAs have been shown to perform essential roles during tissue development and disease. Although roles for microRNAs in the myocardium during embryo development and cardiac disease have been demonstrated, very little is know about their role in the endocardium or during cardiac valve formation. OBJECTIVE: To study the role of microRNAs in cardiac valve formation. METHODS AND RESULTS: We show that zebrafish dicer mutant embryos, lacking mature miRNAs, form excessive endocardial cushions. By screening miRNAs expressed in the heart, we found that miR-23 is both necessary and sufficient for restricting the number of endocardial cells that differentiate into endocardial cushion cells. In addition, in mouse endothelial cells, miR-23 inhibited a transforming growth factor-ß-induced endothelial-to-mesenchymal transition. By in silico screening of expression data with predicted miR-23 target sites combined with in vivo testing, we identified hyaluronic acid synthase 2 (Has2), Icat, and Tmem2 as novel direct targets of miR-23. Finally, we demonstrate that the upregulation of Has2, an extracellular remodeling enzyme required for endocardial cushion and valve formation, is responsible for the excessive endocardial cushion cell differentiation in dicer mutants. CONCLUSIONS: MiR-23 in the embryonic heart is required to restrict endocardial cushion formation by inhibiting Has2 expression and extracellular hyaluronic acid production.

Distinct functions of Mst77F and protamines in nuclear shaping and chromatin condensation during Drosophila spermiogenesis.

Chromatin reorganisation is a major event towards the end of mammalian and Drosophila spermatogenesis. In Drosophila, we previously identified protamine A, protamine B and Mst77F as major chromatin components of the mature sperm. Here, an antibody against Mst77F reveals a dual expression pattern of Mst77F as a chromatin component and in association with microtubules during nuclear shaping. Spermatids of ms(3)nc3 (Mst77F(1)) mutants show disturbed nuclear shaping, instability of perinuclear microtubules but no obvious chromatin condensation defects. Furthermore, we generated a deletion including both protamine genes (prot Delta) and observed that in Drosophila, protamine genes are not haploinsufficient in contrast to those of mice and humans. Moreover, we show that in prot Delta mutants, histone degradation, distribution of DNA breaks and Tpl(94D)-eGFP and Mst77F expression proceed as in wild-type males. Surprisingly, in homozygous prot Delta mutants, males are fertile and sperm are motile, while about 20% of sperm show abnormally shaped nuclei. The latter phenotype can be rescued by supplying protamine-eGFP but not by supplying Mst77F-eGFP. Finally, we demonstrate a 21% increase in X-ray-induced mutation rate of prot Delta sperm. These data support the long-standing hypothesis that the switch from a histone- to protamine-based chromatin protects the paternal genome from mutagens.