Offering prenatal diagnostic tests: European guidelines for clinical practice

For over four decades, it has been possible to offer prenatal diagnostic testing for fetal abnormalities. Prenatal testing is now available for a wide range of monogenic disorders as well as chromosomal abnormalities and should be provided within the ethical framework of informed consent and autonomous choice. However, there are no published guidelines for health professionals from varied disciplines who offer prenatal diagnosis (PND) in a range of possible settings including departments of maternity, obstetrics and clinical genetics. We used an Expert Group technique to develop a set of guidelines for provision of prenatal diagnostic services. Thirteen European health professionals, all experts in PND, participated in a workshop to develop the guidelines, which were then subjected to a wide consultation process. The objective of PND was defined as providing prenatal diagnostic testing services (for genetic conditions) that enable families to make informed choices consistent with their individual needs and values and which support them in dealing with the outcome of such testing. General principles, logistical considerations, clinical care and counselling topics are all described and are equally applicable to invasive and non-invasive testing. These guidelines provide a framework for ethical clinical care; however, they are flexible enough to enable practitioners to adapt them to their particular setting. Ideally, an individualised approach to each family is required to ensure autonomous choice and informed consent regarding prenatal diagnostic testing within the local ethical and legal framework.     

Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study

Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD.     

Separation of on-target efficacy from adverse effects through rational design of a bitopic adenosine receptor agonist

The concepts of allosteric modulation and biased agonism are revolutionizing modern approaches to drug discovery, particularly in the field of G protein-coupled receptors (GPCRs). Both phenomena exploit topographically distinct binding sites to promote unique GPCR conformations that can lead to different patterns of cellular responsiveness. The adenosine A₁ GPCR (A₁AR) is a major therapeutic target for cardioprotection, but current agents acting on the receptor are clinically limited for this indication because of on-target bradycardia as a serious adverse effect. In the current study, we have rationally designed a novel A₁AR ligand (VCP746)—a hybrid molecule comprising adenosine linked to a positive allosteric modulator—specifically to engender biased signaling at the A₁AR. We validate that the interaction of VCP746 with the A₁AR is consistent with a bitopic mode of receptor engagement (i.e., concomitant association with orthosteric and allosteric sites) and that the compound displays biased agonism relative to prototypical A₁AR ligands. Importantly, we also show that the unique pharmacology of VCP746 is (patho)physiologically relevant, because the compound protects against ischemic insult in native A₁AR-expressing cardiomyoblasts and cardiomyocytes but does not affect rat atrial heart rate. Thus, this study provides proof of concept that bitopic ligands can be designed as biased agonists to promote on-target efficacy without on-target side effects.G protein-coupled receptors (GPCRs) are the largest family of cell surface proteins and tractable drug targets (1, 2). Unfortunately, there remains a high attrition rate associated with traditional GPCR-based drug discovery that, in part, reflects an emphasis on the endogenous agonist binding (orthosteric) site as the predominant means of achieving selective GPCR drug targeting (3). Over the last decade, substantial breakthroughs have occurred in the exploitation of topographically distinct GPCR allosteric sites as a means for attaining greater selectivity, especially in those instances where there is high sequence similarity in the orthosteric site across GPCR subtypes (4–6). However, there are increasing examples where both the therapeutic effect and adverse effects are mediated by the same GPCR target (7). In these situations, the desired selectivity needs to be attained at the level of the intracellular signaling pathways linked to a given receptor subtype.GPCRs are highly dynamic proteins, fluctuating between different conformations; these conformations can be linked to different cellular outcomes (8). Thus, chemically distinct ligands, interacting with either orthosteric or allosteric sites, have the potential to stabilize different interaction networks within a GPCR to promote a subset of signaling pathways linked to the receptor at the expense of others. This phenomenon has been termed biased agonism (7, 9, 10). The overall promise of biased agonism is the ability to design GPCR ligands that selectively engage therapeutically relevant signaling pathways while sparing pathways that contribute to undesirable side effects mediated by the same target.The adenosine receptor (AR) family is an important class of physiologically and therapeutically relevant GPCRs that can benefit substantially from more selective drug targeting. Although all four AR subtypes are expressed in the mammalian heart (11, 12), the well-known protective effects of adenosine in this tissue are predominantly mediated by the adenosine A₁ receptor (A₁AR) subtype, especially under conditions of ischemia and reperfusion injury (13–17). Unfortunately, the transition of A₁AR agonists into the clinic has been severely hindered because of high doses causing on-target bradycardia, atrioventricular block, and hypotension (13, 18). As a consequence, clinical trials of AR agonists have had limited success because of the suboptimal dose of agonist that can be used (19–22). It is possible that this problem may be overcome through the exploitation of biased agonism at the A₁AR.Although no study has identified biased orthosteric A₁AR ligands, we recently showed that the 2-amino-3-benzoylthiophene allosteric modulator (VCP171) could promote biased signaling in the activity of the prototypical orthosteric agonist, R(-)N6-(2-phenylisopropyl) adenosine (R-PIA) (23). Thus, we hypothesized that the rational design of a bitopic ligand (i.e., a class of hybrid molecule containing both orthosteric and allosteric pharmacophores) (24–26) may be able to achieve high efficacy and biased agonism at the A₁AR in a single molecule. Herein, we report proof of concept that it is possible to use this approach as a means to dissociate on-target efficacy from on-target side effects.     

Positive and negative allosteric modulators promote biased signaling at the calcium-sensing receptor

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor whose function can be allosterically modulated in a positive or negative manner by calcimimetics or calcilytics, respectively. Indeed, the second-generation calcimimetic, cinacalcet, has proven clinically useful in the treatment of chronic kidney disease patients with secondary hyperparathyroidism but is not widely used in earlier stages of renal disease due to the potential to predispose such patients to hypocalcaemia and hyperphosphatemia. The development of a biased CaSR ligand that is more selective for specific signaling pathway(s) leading only to beneficial effects may overcome this limitation. The detection of such stimulus-bias at a G protein-coupled receptor requires investigation across multiple signaling pathways and the development of methods to quantify the effects of allosteric ligands on orthosteric ligand affinity and cooperativity at each pathway. In the current study, we determined the effects of the calcimimetics, NPS-R568 or cinacalcet, and the calcilytic, NPS-2143, on Ca(o)(2+)-mediated intracellular Ca(2+) mobilization, ERK1/2 phosphorylation, and plasma membrane ruffling in a stably transfected human embryonic kidney 293-TREx c-myc-CaSR cell line and applied a novel analytical model to quantify these modulator effects. We present quantitative evidence for the generation of stimulus bias by both positive and negative allosteric modulators of the CaSR, manifested as greater allosteric modulation of intracellular Ca(2+) mobilization relative to ERK1/2 phosphorylation, and a higher affinity of the modulators for the state of the CaSR mediating plasma membrane ruffling relative to the other two pathways. Our findings provide the first evidence that an allosteric modulator used in clinical practice exhibits stimulus bias.     

Common variation at 6p21.31 (BAK1) influences the risk of chronic lymphocytic leukemia

We performed a meta-analysis of 3 genome-wide association studies to identify additional common variants influencing chronic lymphocytic leukemia (CLL) risk. The discovery phase was composed of genome-wide association study data from 1121 cases and 3745 controls. Replication analysis was performed in 861 cases and 2033 controls. We identified a novel CLL risk locus at 6p21.33 (rs210142; intronic to the BAK1 gene, BCL2 antagonist killer 1; P = 9.47 × 10(-16)). A strong relationship between risk genotype and reduced BAK1 expression was shown in lymphoblastoid cell lines. This finding provides additional support for polygenic inheritance to CLL and provides further insight into the biologic basis of disease development.     

Fractionated doses of gemtuzumab ozogamicin with escalated doses of daunorubicin and cytarabine as first acute myeloid leukemia salvage in patients aged 50-70-year old: a phase 1/2 study of the acute leukemia French association

This Phase 1/2 study aimed to determine optimal doses of daunorubicin (DNR; mg/m²) and cytarabine (mg/m²) to be combined with fractionated doses of gemtuzumab ozogamicin (GO, Mylotarg®; 3 mg/m² on day 1, 4, and 7) satisfying safety requirements. Three dose levels of DNR/AraC were investigated namely (45, 100), (60, 100), and (60, 200). Patients included were acute myeloid leukemia in first relapse, aged 50–70 years. Hematological recovery was 31 days for neutrophil and 32 days for platelet counts. A documented infectious episode > Grade 2 occurred in 11/20 patients (55%). None of the 20 patients had signs of veno‐occlusive disease. Overall, eleven patients reached complete remission (CR), two CR with incomplete platelets recovery. The results showed that combination of fractionated GO doses with DNR at 60 mg/m²/d for 3 days and cytarabine at 200 mg/m²/d for 7 days is tolerable and could be further investigated in the front‐line therapy. Am. J. Hematol., 2012. © 2011 Wiley Periodicals, Inc.     

A structured interview guide for global impressions: increasing reliability and scoring accuracy for CNS trials

Background

The clinical global impression of severity (CGI-S) scale is a frequently used rating instrument for the assessment of global severity of illness in Central Nervous System (CNS) trials. Although scoring guidelines have been proposed to anchor these scores, the collection of sufficient documentation to support the derived score is not part of any standardized interview procedure. It is self evident that the absence of a standardized, documentary format can affect inter-rater reliability and may adversely affect the accuracy of the resulting data.

Method

We developed a structured interview guide for global impressions (SIGGI) and evaluated the instrument in a 2-visit study of ambulatory patients with Major Depressive Disorder (MDD) or schizophrenia. Blinded, site-independent raters listened to audio recorded SIGGI interviews administered by site-based CGI raters. We compared SIGGI-derived CGI-S scores between the two separate site-based raters and the site-independent raters.

Results

We found significant intraclass correlations (p = 0.001) on all SIGGI-derived CGI-S scores between two separate site-based CGI raters with each other (r = 0.768) and with a blinded, site-independent rater (r = 0.748 and r = 0.706 respectively) and significant Pearson’s correlations between CGI-S scores with all MADRS validity comparisons for MDD and PANSS comparisons for schizophrenia (p- 0.001 in all cases). Compared to site-based raters, the site-independent raters gave identical “dual” CGI-S scores to 67.6% and 68.2% of subjects at visit 1 and 77.1% at visit 2.

Conclusion

We suggest that the SIGGI may improve the inter-rater reliability and scoring precision of the CGI-S and have broad applicability in CNS clinical trials.