Improving preimplantation genetic diagnosis (PGD) reliability by selection of sperm donor with the most informative haplotype.

BACKGROUND: The study is aimed to describe a novel strategy that increases the accuracy and reliability of PGD in patients using sperm donation by pre-selecting the donor whose haplotype does not overlap the carrier's one. METHODS: A panel of 4-9 informative polymorphic markers, flanking the mutation in carriers of autosomal dominant/X-linked disorders, was tested in DNA of sperm donors before PGD. Whenever the lengths of donors' repeats overlapped those of the women, additional donors' DNA samples were analyzed. The donor that demonstrated the minimal overlapping with the patient was selected for IVF. RESULTS: In 8 out of 17 carriers the markers of the initially chosen donors overlapped the patients' alleles and 2-8 additional sperm donors for each patient were haplotyped. The selection of additional sperm donors increased the number of informative markers and reduced misdiagnosis risk from 6.00% ± 7.48 to 0.48% ±0.68. The PGD results were confirmed and no misdiagnosis was detected. CONCLUSIONS: Our study demonstrates that pre-selecting a sperm donor whose haplotype has minimal overlapping with the female's haplotype, is critical for reducing the misdiagnosis risk and ensuring a reliable PGD. This strategy may contribute to prevent the transmission of affected IVF-PGD embryos using a simple and economical procedure. TRIAL REGISTRATION: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. DNA testing of donors was approved by the institutional Helsinki committee (registration number 319-08TLV, 2008). The present study was approved by the institutional Helsinki committee (registration number 0385-13TLV, 2013).

Complex chromosomal rearrangement-a lesson learned from PGS.

PURPOSE: The aim of the study is to report a case of non-diagnosed complex chromosomal rearrangement (CCR) identified by preimplantation genetic screening (PGS) followed by preimplantation genetic diagnosis (PGD) which resulted in a pregnancy and delivery of healthy offspring. METHODS: A 29-year-old woman and her spouse, both diagnosed previously with normal karyotypes, approached our IVF-PGD center following eight early spontaneous miscarriages. PGS using chromosomal microarray analysis (CMA) was performed on biopsied trophectoderm. Fluorescence in situ hybridization (FISH), as well as re-karyotype, were performed on metaphase derived from peripheral blood of the couple. Subsequently, in the following PGD cycle, a total of seven blastocysts underwent CMA. RESULTS: A gain or loss at three chromosomes (3, 7, 9) was identified in six out of seven embryos in the first PGS-CMA cycle. FISH analysis of parental peripheral blood samples demonstrated that the male is a carrier of a CCR involving those chromosomes; this was in spite of a former diagnosis of normal karyotypes for both parents. Re-karyotype verified the complex translocation of 46,XY,t (3;7;9)(q23;q22;q22). Subsequently, in the following cycle, a total of seven blastocysts underwent PGD-CMA for the identified complex translocation. Two embryos were diagnosed with balanced chromosomal constitution. A single balanced embryo was transferred and pregnancy was achieved, resulting in the birth of a healthy female baby. CONCLUSIONS: PGS employing CMA is an efficient method to detect unrevealed chromosomal abnormalities, including complicated cases of CCR. The combined application of array CGH and FISH technologies enables the identification of an increased number of CCR carriers for which PGD is particularly beneficial.

Recruitment of Gßγ controls the basal activity of G-protein coupled inwardly rectifying potassium (GIRK) channels: crucial role of distal C terminus of GIRK1.

The G-protein coupled inwardly rectifying potassium (GIRK, or Kir3) channels are important mediators of inhibitory neurotransmission via activation of G-protein coupled receptors (GPCRs). GIRK channels are tetramers comprising combinations of subunits (GIRK1-4), activated by direct binding of the Gßγ subunit of Gi/o proteins. Heterologously expressed GIRK1/2 exhibit high, Gßγ-dependent basal currents (Ibasal) and a modest activation by GPCR or coexpressed Gßγ. Inversely, the GIRK2 homotetramers exhibit low Ibasal and strong activation by Gßγ. The high Ibasal of GIRK1 seems to be associated with its unique distal C terminus (G1-dCT), which is not present in the other subunits. We investigated the role of G1-dCT using electrophysiological and fluorescence assays in Xenopus laevis oocytes and protein interaction assays. We show that expression of GIRK1/2 increases the plasma membrane level of coexpressed Gßγ (a phenomenon we term 'Gßγ recruitment') but not of coexpressed Gαi3. All GIRK1-containing channels, but not GIRK2 homomers, recruited Gßγ to the plasma membrane. In biochemical assays, truncation of G1-dCT reduces the binding between the cytosolic parts of GIRK1 and Gßγ, but not Gαi3. Nevertheless, the truncation of G1-dCT does not impair activation by Gßγ. In fluorescently labelled homotetrameric GIRK1 channels and in the heterotetrameric GIRK1/2 channel, the truncation of G1-dCT abolishes Gßγ recruitment and decreases Ibasal. Thus, we conclude that G1-dCT carries an essential role in Gßγ recruitment by GIRK1 and, consequently, in determining its high basal activity. Our results indicate that G1-dCT is a crucial part of a Gßγ anchoring site of GIRK1-containing channels, spatially and functionally distinct from the site of channel activation by Gßγ.

Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by GalphaiGDP and Gbetagamma.

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gbetagamma following activation of Gi/o proteins via G protein-coupled receptors (GPCRs). Although Galphai subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Galphai3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gbegagamma. Here we show that this regulation is exerted by the 'inactive' GDP-bound Galphai3GDP and involves the formation of Galphai3betagamma heterotrimers, by a mechanism distinct from mere sequestration of Gbetagamma 'away' from the channel. The regulation of basal and Gbetagamma-evoked current was produced by the 'constitutively inactive' mutant of Galphai3, Galphai3G203A, which strongly binds Gbetagamma, but not by the 'constitutively active' mutant, Galphai3Q204L, or by Gbetagamma-scavenging proteins. Furthermore, regulation by Galphai3G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gbetagamma enhanced the binding of Galphai3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gbetagamma effector, showing low Ibasal and strong Gbetagamma-dependent activation. Expression of Galphai3G203A did not affect either Ibasal or Gbetagamma-induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gbegagamma. Expression of Galphai3GDP reduced Ibasal and restored the ability of Gbetagamma to activate GIRK1*, like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1*. These results demonstrate that GIRK1 containing channels are regulated by both Galphai3GDP and Gbetagamma, while GIRK2 is a Gbetagamma-effector insensitive to Galphai3GDP.

G(alpha)(i) controls the gating of the G protein-activated K(+) channel, GIRK.

GIRK (Kir3) channels are activated by neurotransmitters coupled to G proteins, via a direct binding of G(beta)(gamma). The role of G(alpha) subunits in GIRK gating is elusive. Here we demonstrate that G(alpha)(i) is not only a donor of G(beta)(gamma) but also regulates GIRK gating. When overexpressed in Xenopus oocytes, GIRK channels show excessive basal activity and poor activation by agonist or G(beta)(gamma). Coexpression of G(alpha)(i3) or G(alpha)(i1) restores the correct gating parameters. G(alpha)(i) acts neither as a pure G(beta)(gamma) scavenger nor as an allosteric cofactor for G(beta)(gamma). It inhibits only the basal activity without interfering with G(beta)(gamma)-induced response. Thus, GIRK is regulated, in distinct ways, by both arms of the G protein. G(alpha)(i) probably acts in its GDP bound form, alone or as a part of G(alpha)(beta)(gamma) heterotrimer.

G protein-activated K+ channels: a reporter for rapid activation of G proteins by lysophosphatidic acid in Xenopus oocytes.

Threshold concentrations of lysophosphatidic acid (LPA) or acetylcholine (ACh) induce pertussis toxin (PTX)-sensitive rapid desensitization of responses to LPA in Xenopus oocytes. To demonstrate that threshold [LPA] rapidly activates Gi/o proteins, we used the G protein-activated K+ channel (GIRK) as a reporter. Low [LPA] induced IK+ in <3 s of the agonist addition with little or no activation of chloride current. Depletion of Galphao/Galphao1 each decreased the LPA-induced IK+ by approximately 40-50%, while PTX completely abolished it. This is the first direct evidence showing the activation of GIRK by LPA, and the involvement of G proteins of the Go family in rapid desensitization of LPA responses.

Galphai3 primes the G protein-activated K+ channels for activation by coexpressed Gbetagamma in intact Xenopus oocytes.

G protein-activated K+ channels (GIRK) mediate postsynaptic inhibitory effects of neurotransmitters in the atrium and in the brain by coupling to G protein-coupled receptors (GPCRs). In neurotransmitter-dependent GIRK signalling, Gbetagamma is released from the heterotrimeric Galphabetagamma complex upon GPCR activation, activating the channel and attenuating its rectification. Now it becomes clear that Galpha is more than a mere Gbetagamma donor. We have proposed that Galphai3-GDP regulates GIRK gating, keeping its basal activity low but priming (predisposing) the channel for activation by agonist in intact cells, and by Gbetagamma in excised patches. Here we have further investigated GIRK priming by Galphai3 using a model in which the channel was activated by coexpression of Gbetagamma, and the currents were measured in intact Xenopus oocytes using the two-electrode voltage clamp technique. This method enables the bypass of GPCR activation during examination of the regulation of the channel in intact cells. Using this method, we further characterize the priming phenomenon. We tested and excluded the possibility that our estimates of priming are affected by artifacts caused by series resistance or large K+ fluxes. We demonstrate that both Galphai3 and membrane-attached Gbetagamma scavenger protein, m-phosducin, reduce the basal channel activity. However, Galphai3 allows robust channel activation by coexpressed Gbetagamma, in sharp contrast to m-phosducin, which causes a substantial reduction in the total Gbetagamma-induced current. Furthermore, Galphai3 also does not impair the Gbetagamma-dependent attenuation of the channel rectification, in contrast to m-phosducin, which prevents this Gbetagamma-induced modulation. The Galphai3-induced enhancement of direct activation of GIRK by Gbetagamma, demonstrated here for the first time in intact cells, strongly supports the hypothesis that Galphai regulates GIRK gating under physiological conditions.

Galphai1 and Galphai3 differentially interact with, and regulate, the G protein-activated K+ channel.

G protein-activated K(+) channels (GIRKs; Kir3) are activated by direct binding of Gbetagamma subunits released from heterotrimeric G proteins. In native tissues, only pertussis toxin-sensitive G proteins of the G(i/o) family, preferably Galpha(i3) and Galpha(i2), are donors of Gbetagamma for GIRK. How this specificity is achieved is not known. Here, using a pull-down method, we confirmed the presence of Galpha(i3-GDP) binding site in the N terminus of GIRK1 and identified novel binding sites in the N terminus of GIRK2 and in the C termini of GIRK1 and GIRK2. The non-hydrolyzable GTP analog, guanosine 5'-3-O-(thio)triphosphate, reduced the binding of Galpha(i3) by a factor of 2-4. Galpha(i1-GDP) bound to GIRK1 and GIRK2 much weaker than Galpha(i3-GDP). Titrated expression of components of signaling pathway in Xenopus oocytes and their activation by m2 muscarinic receptors revealed that G(i3) activates GIRK more efficiently than G(i1), as indicated by larger and faster agonist-evoked currents. Activation of GIRK by purified Gbetagamma in excised membrane patches was strongly augmented by coexpression of Galpha(i3) and less by Galpha(i1). Differences in physical interactions of GIRK with GDP-bound Galpha subunits, or Galphabetagamma heterotrimers, may dictate different extents of Galphabetagamma anchoring, influence the efficiency of GIRK activation by Gbetagamma, and play a role in determining signaling specificity.