Performance and Diagnostic Value of Genome-Wide Noninvasive Prenatal Testing in Multiple Gestations.

OBJECTIVE: To evaluate the accuracy and diagnostic value of genome-wide noninvasive prenatal testing (NIPT) for the detection of fetal aneuploidies in multiple gestations, with a focus on dichorionic-diamniotic twin pregnancies. METHODS: We performed a retrospective cohort study including data from pregnant women with a twin or higher-order gestation who underwent genome-wide NIPT at one of the eight Belgian genetic centers between November 1, 2013, and March 1, 2020. Chorionicity and amnionicity were determined by ultrasonography. Follow-up invasive testing was carried out in the event of positive NIPT results. Sensitivity and specificity were calculated for the detection of trisomy 21, 18, and 13 in the dichorionic-diamniotic twin cohort. RESULTS: Unique NIPT analyses were performed for 4,150 pregnant women with a multiple gestation and an additional 767 with vanishing gestations. The failure rate in multiple gestations excluding vanishing gestations ranged from 0% to 11.7% among the different genetic centers. Overall, the failure rate was 4.8%, which could be reduced to 1.2% after single resampling. There were no common fetal trisomies detected among the 86 monochorionic-monoamniotic and 25 triplet cases. Two monochorionic-diamniotic twins had an NIPT result indicative of a trisomy 21, which was confirmed in both fetuses. Among 2,716 dichorionic-diamniotic twin gestations, a sensitivity of 100% (95% CI 74.12-100%) and a specificity of 100% (95% CI 99.86-100%) was reached for trisomy 21 (n=12). For trisomy 18 (n=3), the respective values were 75% (95% CI 30.06-95.44%) sensitivity and 100% (95% CI 99.86-100%) specificity, and for trisomy 13 (n=2), 100% (95% CI 20.65-100%) sensitivity and 99.96% (95% CI 99.79-99.99%) specificity. In the vanishing gestation group, 28 NIPT results were positive for trisomy 21, 18, or 13, with only five confirmed trisomies. CONCLUSION: Genome-wide NIPT performed accurately for detection of aneuploidy in dichorionic-diamniotic twin gestations

Interaction between polycystin-2 and the inositol 1,4,5-trisphosphate receptor and its function in the endoplasmic reticulum

Interaction between polycystin-2 and the inositol 1,4,5-trisphosphate receptor and their function in the endoplasmic reticulum Autosomal dominant polycystic kidney disease (ADPKD) is an inherited human disorder that affects more than six million people worldwide and that is the most common monogenic cause of kidney failure in humans. ADPKD results in end-stage renal disease in ~50% of the affected individuals by the age of 60. ADPKD arises as a consequence of mutations oftwo genes PKD1 and PKD2 , encoding the integral membrane proteins polycystin-1 (PKD1, ~460 kDa) and polycystin-2 (TRPP2, ~110 kDa) respectively, resulting in a disturbance in intracellular Ca2+ signaling .Most mutations identified in affected families appear to truncate and (or) inactivate either of both proteins. TRPP2 is a 968-amino-acid protein with six predicted transmembrane domains and is highly conserved amongmulticellular organisms and widely expressed in various tissues. There is a long-standing debate on the subcellular localization of TRPP2. TRPP2 has been detected (i) in the plasma membrane, where it is supposed to form a receptor-operated, non-selective cation channel, (ii) in the primary cilium, where it could act as a mechanosensitive channel, possibly in association with PKD1, TRPC1 or TRPV4, (iii) in the endoplasmic reticulum (ER), where it is proposed to function as an intracellular Ca2+-release channel, but also in centrosomes and in mitotic spindles ofdividing cells. However, the predominant subcellular localization of TRPP2 is in the ER, as shown by sensitivity to endoglycosidase H, immunofluorescence, co-localization and co-distribution with ER-resident proteins. Interaction between TRPP2 and the two major intracellular Ca2+-release channels, the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor (IP3R), has been reported. However, the exact mechanism throughwhich polycystins in general and TRPP2 in particular modulate intracellular Ca2+ signaling is not yet understood. Therefore we have performed adetailed analysis of the molecular and functional relation between the IP3R and TRPP2. Here, w e identified the molecular determinants of the interaction between TRPP2 and the IP3R, the major intracellular Ca2+ channel in the ER. GST pull-down experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP3R as directly responsible for the interaction. In order to investigate the functionalrelevance of TRPP2 in the ER, we r e-introduced the protein in renal epithelial cells from TRPP2-/- mouse using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca2+ release in intact cells and IP3-induced Ca2+ release in permeabilized cells. Using pathological mutants of TRPP2 (R740X and D509V)and competing peptides, we demonstrated that TRPP2 amplified the Ca2+ signal by a local Ca2+-induced Ca2+-release (CICR) mechanism, which only occurred in the presence of the TRPP2/IP3R interaction, and not via altered IP3R-channel activity. An additional support for the importance of aclose interaction between TRPP2 and the IP3R stems from the observationthat a global rise in free cytosolic [Ca2+] as provoked by ionomycin orthapsigargin did not result in an increased TRPP2-induced Ca2+ signal. Moreover, our results indicate that this interaction was instrumental inthe formation of Ca2+ microdomains necessary for initiating CICR. Therefore we propose a model for the physiological mechanism behind the potentiated Ca2+ release related to TRPP2 activation in the ER. Upon cell stimulation, IP3 is produced and activates the IP3R which leads to a local rise in free cytosolic [Ca2+] that subsequently can activate the TRPP2 channel as a CICR channel. The data strongly suggest that defects in this mechanism may account for the altered Ca2+ signalingassociated with pathological TRPP2 mutations and therefore contribute to the development of ADPKD.TABLE OF CONTENTS Introduction 1 1. Autosomal dominant polycystic kidney disease 1 2. Polycystin-1 6 3. Polycystin-2 9 3.1. Subcellular localization and function 10 3.1.1. Function at the plasma membrane 11 3.1.2. Function at the primary cilium 13 3.1.3. Function at the endoplasmic reticulum 16 3.2. Regulation of the trafficking of polycystin-2 17 3.3. Activation mechanisms 18 4. Signal-transduction pathways involving polycystins 21 4.1. The janus kinase-signal transducers and activator of transcription pathway 21 4.2. The inhibitor of DNA binding pathway 22 4.3. The mammalian-target-of-rapamycin pathway 23 4.4. Intracellular Ca2+ signaling and cAMP signaling 24 5. Therapeutic prospects in autosomal dominant polycystic kidney disease 25 6. Ca2+ signaling 28 7. Spatiotemporal aspects of Ca2+ signaling 29 8. The inositol 1,4,5-trisphosphate receptor 31 8.1. General considerations 31 8.2. Structure-function relations 32 8.2.1. Ligand-binding domain 32 8.2.2. C-terminal domain: the pore and the gate 33 8.3. Regulation 34 Aims of the study 36 1. General aim 36 2. Objectives 36 Materials & Methods 38 1. Materials 38 2. Molecular and biochemical techniques 38 2.1. DNA constructs and adenovirus construction 38 2.2. Cell culture 40 2.3. Antibodies 40 2.4. Immunoprecipitation 41 2.5. Preparation of GST- or HIS-fusion proteins 41 2.6. GST-pull down 42 2.7. SDS-PAGE and Western blotting 42 2.8. Confocal imaging 43 2.9. RNA interference 43 3. Functional measurements 44 3.1. [³H]IP3 binding 44 3.2. [Ca2+] measurements 44 3.3. In vitro phosphorylation and dephosphorylation of polycystin-2 45 Results & Discussion 47 1. Molecular analysis of the interaction between polycystin-2 and the inositol 1,4,5-trisphosphate receptor 47 1.1. Full-length polycystin-2 interacts with full-length inositol 1,4,5-trisphosphate receptor type 1 or 3 47 1.2. Interaction between polycystin-2 and the N-terminal ligand-binding domain of the inositol 1,4,5-trisphosphate receptor 48 1.3. A positively charged cluster in the suppressor region of the ligand-binding domain of the inositol 1,4,5-trisphosphate receptor interacts with an acidic cluster in the C-terminal cytoplasmic tail of polycystin-2 50 1.4. The C-terminal cytoplasmic tail of polycystin-2 partially inhibits the binding of inositol 1,4,5-trisphosphate to the ligand-binding domain of the inositol 1,4,5-trisphosphate receptor through binding to the suppressor region 55 2. Functional analysis of the effect of the interaction between polycystin-2 and the inositol 1,4,5-trisphosphate receptor on Ca2+ signaling 56 2.1. Polycystin-2 potentiates ATP-induced intracellular Ca2+ release in intact cells 56 2.2. Polycystin-2 potentiates inositol 1,4,5-trisphosphate-induced Ca2+ release in permeabilized cells 59 2.3. Stimulation of inositol 1,4,5-trisphosphate-induced Ca2+ release is dependent on polycystin-2 function and on interaction with the inositol 1,4,5-trisphosphate receptor 63 3. Regulation of polycystin-2 by (de)phosphorylation 67 3.1. S810, located in an acidic cluster in the C-terminal cytoplasmic tail of polycystin-2, is phosphorylated by casein kinase 2 68 3.2. Protein phosphatase 2A specifically dephosphorylates polycystin-2 in vitro 69 3.3. Polycystin-2 specifically interacts with PR130/B” 70 3.4. Effect of dephosphorylation on polycystin-2 function 71 4. Proposed model 72 General conclusions 74 Future perspectives 78 1. Regulation of polycystin-2 by (de)phosphorylation 78 2. Is cAMP elevation in autosomal dominant polycystic kidney disease linked to polycystin-2-mediated Ca2+ signaling? 78 Summary 80 Samenvatting 82 References 84 Curriculum vitae 95 1. List of publications 95 2. List of abstracts 96 3. Participation in international meetings 97nrpages: 110status: publishe

The IRBIT domain adds new functions to the AHCY family

During the past few years, the IRBIT domain has emerged as an important add-on of S-adenosyl-L-homocystein hydrolase (AHCY), thereby creating the new family of AHCY-like proteins. In this review, we discuss the currently available data on this new family of proteins. We describe the IRBIT domain as a unique part of these proteins and give an overview of its regulation via (de)phosphorylation and proteolysis. The second part of this review is focused on the potential functions of the AHCY-like proteins. We propose that the IRBIT domain serves as an anchor for targeting AHCY-like proteins towards cytoplasmic targets. This leads to regulation of (i) intracellular Ca2+ via the inositol 1,4,5-trisphosphate receptor (IP3R), (ii) intracellular pH via the Na+/HCO3- cotransporters (NBCs); whereas inactivation of the IRBIT domain induces (iii) nuclear translocation and regulation of AHCY activity. Dysfunction of AHCY-like proteins will disturb these three important functions, with various biological implications.status: publishe

Polycystin-2 activation by inositol 1,4,5-trisphosphate-induced Ca2+ release requires its direct association with the inositol 1,4,5-trisphosphate receptor in a signaling microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca2+ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca2+ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP3R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2(-/-) mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca2+ release in intact cells and IP3-induced Ca2+ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca2+ signal by a local Ca2+-induced Ca2+-release mechanism, which only occurred in the presence of the TRPP2-IP3R interaction, and not via altered IP3R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca2+ microdomains necessary for initiating Ca2+-induced Ca2+ release. The data strongly suggest that defects in this mechanism may account for the altered Ca2+ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.status: publishe

F508del-CFTR increases intracellular Ca(2+) signaling that causes enhanced calcium-dependent Cl(-) conductance in cystic fibrosis

In many cells, increase in intracellular calcium ([Ca(2+)](i)) activates a Ca(2+)-dependent chloride (Cl(-)) conductance (CaCC). CaCC is enhanced in cystic fibrosis (CF) epithelial cells lacking Cl(-) transport by the CF transmembrane conductance regulator (CFTR). Here, we show that in freshly isolated nasal epithelial cells of F508del-homozygous CF patients, expression of TMEM16A and bestrophin 1 was unchanged. However, calcium signaling was strongly enhanced after induction of expression of F508del-CFTR, which is unable to exit the endoplasmic reticulum (ER). Since receptor-mediated [Ca(2+)](i) increase is Cl(-) dependent, we suggested that F508del-CFTR may function as an ER chloride counter-ion channel for Ca(2+). This was confirmed by expression of the double mutant F508del/G551D-CFTR, which remained in the ER but had no effects on [Ca(2+)](i). Moreover, F508del-CFTR could serve as a scavenger for inositol-1,4,5-trisphosphate [IP3] receptor binding protein released with IP(3) (IRBIT). Our data may explain how ER-localized F508del-CFTR controls intracellular Ca(2+) signaling.status: publishe

Polycystin-1 and polycystin-2 are both required to amplify inositol-trisphosphate-induced Ca2+ release

Autosomal dominant polycystic kidney disease is caused by loss-of-function mutations in the PKD1 or PKD2 genes encoding respectively polycystin-1 and polycystin-2. Polycystin-2 stimulates the inositol trisphosphate (IP(3)) receptor (IP(3)R), a Ca(2+)-release channel in the endoplasmic reticulum (ER). The effect of ER-located polycystin-1 is less clear. Polycystin-1 has been reported both to stimulate and to inhibit the IP(3)R. We now studied the effect of polycystin-1 and of polycystin-2 on the IP(3)R activity under conditions where the cytosolic Ca(2+) concentration was kept constant and the reuptake of released Ca(2+) was prevented. We also studied the interdependence of the interaction of polycystin-1 and polycystin-2 with the IP(3)R. The experiments were done in conditionally immortalized human proximal-tubule epithelial cells in which one or both polycystins were knocked down using lentiviral vectors containing miRNA-based short hairpins. The Ca(2+) release was induced in plasma membrane-permeabilized cells by various IP(3) concentrations at a fixed Ca(2+) concentration under unidirectional (45)Ca(2+)-efflux conditions. We now report that knock down of polycystin-1 or of polycystin-2 inhibited the IP(3)-induced Ca(2+) release. The simultaneous presence of the two polycystins was required to fully amplify the IP(3)-induced Ca(2+) release, since the presence of polycystin-1 alone or of polycystin-2 alone did not result in an increased Ca(2+) release. These novel findings indicate that ER-located polycystin-1 and polycystin-2 operate as a functional complex. They are compatible with the view that loss-of-function mutations in PKD1 and in PKD2 both cause autosomal dominant polycystic kidney disease.status: publishe

Unraveling the role of polycystin-2/inositol 1,4,5-trisphosphate receptor interaction in Ca2+ signaling

Autosomal dominant polycystic kidney disease (ADPKD) arises as a consequence of mutations of the genes PKD1 and PKD2, encoding respectively the integral membrane proteins polycystin-1 and polycystin-2 (TRPP2), resulting in a disturbance in intracellular Ca2+ signaling. Previously we investigated the interaction between TRPP2 and the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), an intracellular Ca2+ channel in the endoplasmic reticulum (ER). We identified the molecular determinants of this interaction and observed an enhanced IP3-induced Ca2+ release (IICR). Since we found that TRPP2 strongly bound to a cluster of positively charged amino acids in the N-terminal ligand-binding domain (LBD) of the IP3R, we now investigated whether TRPP2 would interfere with the binding of IP3 to the IP3R. In in vitro experiments we observed that TRPP2 partially inhibited the binding of IP3 to the LBD of the IP3R with an IC50 of ~350 nM. The suppressor domain, i.e. the N-terminal 225 amino acids of the LBD of the IP3R, mediated this inhibitory effect of TRPP2 on IP3 binding. The observation that the interaction between the IP3R and TRPP2 decreased IP3 binding is in apparent contrast to the increased IICR. The data can be explained however by a subsequent activation of Ca2+-induced Ca2+ release (CICR) via TRPP2. Implications of this mechanism for cellular Ca2+ signaling are discussed in this addendum