Audit of the first > 7500 noninvasive prenatal aneuploidy tests in a Swiss genetics center.

OBJECTIVES: Noninvasive prenatal testing (NIPT) is actually the most accurate method of screening for fetal chromosomal aberration (FCA). We used pregnancy outcome record to evaluate a complete data set of single nucleotide polymorphism-based test results performed by a Swiss genetics center. MATERIALS AND METHODS: The Panorama® test assesses the risk of fetal trisomies (21, 18 and 13), gonosomal aneuploidy (GAN), triploidy or vanishing twins (VTT) and five different microdeletions (MD). We evaluated all 7549 test results meeting legal and quality requirements taken in women with nondonor singleton pregnancies between April 2013 and September 2016 classifying them as high or low risk. Follow-up ended after 9 months, data collection 7 months later. RESULTS: The Panorama® test provided conclusive results in 96.1% of cases, detecting 153 FCA: T21 n = 76, T18 n = 19, T13 n = 15, GAN n = 19, VTT n = 13 and MD n = 11 (overall prevalence 2.0%). Pregnancy outcome record was available for 68.6% of conclusive laboratory results, including 2.0% high-risk cases. In this cohort the Panorama® test exhibited 99.90% sensitivity for each trisomy; specificity was 99.90% for T21, 99.98% for T18 and 99.94% for T13. False positive rate was 0.10% for T21, 0.02% for T18 and 0.06% for T13. CONCLUSION: SNP-based testing by a Swiss genetics center confirms the expected accuracy of NIPT in FCA detection.

Collagen VI-Related Myopathy Caused by Compound Heterozygous Mutations of COL6A3 in a Consanguineous Kurdish Family.

ABSTRACT: Collagen VI-related myopathies are caused by mutations of COL6A1, COL6A2, and COL6A3 and present with a wide phenotypic spectrum ranging from severe Ulrich congenital muscular dystrophy to mild Bethlem myopathy. Here, we report a consanguineous Kurdish family with 3 siblings affected by autosomal-recessive Bethlem myopathy caused by compound heterozygous mutations of COL6A3. We found the previously described missense mutation c.7447A > G/p.(Lys2483Glu) and a novel large deletion encompassing the exon 1-39 of the COL6A3 gene. Apart from the classical clinical symptoms, all patients had keratoconus, which expands the phenotype of the collagen VI-related myopathies.

Induction of myocardial infarction in adult zebrafish using cryoinjury.

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction(1). By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life(2,3). It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues(4,5). To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries. In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury(6,7). The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex(2,3). Recently, several groups have established the cryoinjury technique in adult zebrafish(8-10). This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species. Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%. To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

The regenerative capacity of the zebrafish heart is dependent on TGFß signaling.

Mammals respond to a myocardial infarction by irreversible scar formation. By contrast, zebrafish are able to resolve the scar and to regenerate functional cardiac muscle. It is not known how opposing cellular responses of fibrosis and new myocardium formation are spatially and temporally coordinated during heart regeneration in zebrafish. Here, we report that the balance between the reparative and regenerative processes is achieved through Smad3-dependent TGFß signaling. The type I receptor alk5b (tgfbr1b) is expressed in both fibrotic and cardiac cells of the injured heart. TGFß ligands are locally induced following cryoinjury and activate the signaling pathway both in the infarct area and in cardiomyocytes in the vicinity of the trauma zone. Inhibition of the relevant type I receptors with the specific chemical inhibitor SB431542 qualitatively altered the infarct tissue and completely abolished heart regeneration. We show that transient scar formation is an essential step to maintain robustness of the damaged ventricular wall prior to cardiomyocyte replacement. Taking advantage of the reversible action of the inhibitor, we dissected the multifunctional role of TGFß signaling into three crucial processes: collagen-rich scar deposition, Tenascin C-associated tissue remodeling at the infarct-myocardium interface, and cardiomyocyte proliferation. Thus, TGFß signaling orchestrates the beneficial interplay between scar-based repair and cardiomyocyte-based regeneration to achieve complete heart regeneration.

The zebrafish heart regenerates after cryoinjury-induced myocardial infarction.

BACKGROUND: In humans, myocardial infarction is characterized by irreversible loss of heart tissue, which becomes replaced with a fibrous scar. By contrast, teleost fish and urodele amphibians are capable of heart regeneration after a partial amputation. However, due to the lack of a suitable infarct model, it is not known how these animals respond to myocardial infarction. RESULTS: Here, we have established a heart infarct model in zebrafish using cryoinjury. In contrast to the common method of partial resection, cryoinjury results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts. As in mammals, the initial stages of the injury response include thrombosis, accumulation of fibroblasts and collagen deposition. However, at later stages, cardiac cells can enter the cell cycle and invade the infarct area in zebrafish. In the subsequent two months, fibrotic scar tissue is progressively eliminated by cell apoptosis and becomes replaced with a new myocardium, resulting in scarless regeneration. We show that tissue remodeling at the myocardial-infarct border zone is associated with accumulation of Vimentin-positive fibroblasts and with expression of an extracellular matrix protein Tenascin-C. Electrocardiogram analysis demonstrated that the reconstitution of the cardiac muscle leads to the restoration of the heart function. CONCLUSIONS: We developed a new cryoinjury model to induce myocardial infarction in zebrafish. Although the initial stages following cryoinjury resemble typical healing in mammals, the zebrafish heart is capable of structural and functional regeneration. Understanding the key healing processes after myocardial infarction in zebrafish may result in identification of the barriers to efficient cardiac regeneration in mammals.

IGF signaling between blastema and wound epidermis is required for fin regeneration.

In mammals, the loss of a limb is irreversible. By contrast, urodele amphibians and teleost fish are capable of nearly perfect regeneration of lost appendages. This ability depends on direct interaction between the wound epithelium and mesenchymal progenitor cells of the blastema. It has been known for decades that contact between the wound epithelium and the underlying blastema is essential for successful regeneration. However, the underlying mechanisms are poorly understood. Here, we show that upon amputation the blastema induces expression of the ligand Igf2b, which then activates IGF signaling specifically in cells of the adjacent apical epithelium. Inhibition of IGF signaling by either morpholino antisense technology, or by specific chemical inhibitors of Igf1 receptor function NVP-AEW541 and NVP-ADW742, impairs fin regeneration. At the cellular level, this block in regeneration is reflected by a lack of the distinctive basal epithelium, increased apoptosis in the wound epidermis and reduced proliferation of blastema cells. Furthermore, induction of the blastemal and wound epidermal markers cannot be supported in the absence of IGF signaling. These data provide evidence that Igf2b expressed in the blastema promotes the properties of the adjacent wound epidermis, which subsequently are necessary for blastema function. Thus, IGF signaling upregulated upon fin amputation represents a signal from the blastema to the wound epithelium, a crucial step in appendage regeneration.