Next-generation sequencing-based genome diagnostics across clinical genetics centers: Implementation choices and their effects

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care

Prenatal stress in pigs

Studies in many species, including humans, have demonstrated that stress during gestation can have long-term developmental, neuroendocrine, and behavioural effects on the offspring. Because pregnant sows can be subjected to regular stressful situations, it is relevant to study whether prenatal stress also affects their piglets. Glucocorticoids play an important role in affecting the fetus during prenatal stress, and therefore, a model to elevate maternal cortisol concentrations during gestation was developed. Main objectives of the project described in this thesis were to determine whether (1) piglet characteristics (e.g. growth, behaviour) are affected by elevated maternal cortisol concentrations during gestation; (2) the timing of elevated maternal cortisol concentrations during gestation is relevant for the observed effects; (3) social challenges for pregnant sows also affect the piglets. Sows orally received hydrocortisone acetate (HCA) during one of three periods (P1, P2, or P3) of gestation. They had elevated salivary cortisol concentrations during the period of HCA administration. Main findings of the study were that piglets from sows that received HCA during any of the three periods had lower birth weights and remained lighter until weaning. During two behavioural tests (the backtest and the tonic immobility test), the number of vocalisations did not differ between HCA male and female piglets, while control males vocalised significantly more than control females. During a novel environment test, P1 and P3 piglets walked more than control piglets. When introduced to an unfamiliar piglet, P1, P2, and P3 male piglets had less non-aggressive encounters than control male piglets. Piglets from the control, P1, and P3 group had less aggressive encounters during the second part of the mixing test, whereas P2 piglets continued fighting. Furthermore, the salivary cortisol concentration in response to i.m. ACTH was lower in female P1 and P3 piglets compared to control piglets. These P1 and P3 females also displayed an elevated temperature response to i.v. LPS injection compared to control females. At slaughter, HCA treatment indirectly decreased lean meat percentage and increased the fat percentage. These results indicate that elevated maternal cortisol concentrations during gestation do affect piglet development, neuroendocrine activity, and behaviour postanatally, and that effects may differ between male and female piglets. In addition, effects depend on the period of gestation during which cortisol concentrations of the sow were elevated. In a social-challenge experiment, pregnant sows were introduced to an unfamiliar sow twice weekly during the last month of gestation. Though these Mix sows gained less weight during gestation and lost less weight during lactation, their salivary cortisol concentrations in response to mixing did not differ from those of (undisturbed) Control sows. Piglets did not differ in birth weight, body weight at weaning, behaviour, nor did their adrenocortical response to ACTH, indicating that regular mixing of pregnant sows at the end of gestation does not affect their offspring. In a second social-challenge experiment, pregnant sows were housed in social groups throughout gestation to determine whether the social rank of pregnant sows during gestation would affect their piglets. Sows were classified as High Social Ranking (HSR) or Low Social Ranking (LSR). Prior to insemination, HSR and LSR sows did not differ in body weight, but HSR sows gained more weight during gestation and, subsequently, lost more weight during lactation. Maternal salivary cortisol concentrations during gestation did not differ between HSR and LSR sows, nor did gestation length, litter size, or percentage of live born piglets. HSR piglets tended to weigh more at birth, and weighed more at weaning. During a novel object test, HSR piglets moved and vocalised more than LSR piglets. In addition, the latency time to touch the novel object was shorter in HSR offspring, and HSR males spent more time near the object than LSR males. At slaughter, HSR offspring had more lean meat than LSR offspring. These results indicate that the sow’s social rank during gestation does affect her own weight gain and loss, and her offspring’s growth and behaviour. Taken together, the results of the experiments described in this thesis demonstrate that both elevated maternal cortisol concentrations and the social rank of the pregnant sow during gestation can affect piglet growth, behaviour, and physiology

Signs of Mood and Anxiety Disorders in Chimpanzees

Background: In humans, traumatic experiences are sometimes followed by psychiatric disorders. In chimpanzees, studies have demonstrated an association between traumatic events and the emergence of behavioral disturbances resembling posttraumatic stress disorder (PTSD) and depression. We addressed the following central question: Do chimpanzees develop posttraumatic symptoms, in the form of abnormal behaviors, which cluster into syndromes similar to those described in human mood and anxiety disorders? Methodology/Principal Findings: In phase 1 of this study, we accessed case reports of chimpanzees who had been reportedly subjected to traumatic events, such as maternal separation, social isolation, experimentation, or similar experiences. We applied and tested DSM-IV criteria for PTSD and major depression to published case reports of 20 chimpanzees identified through PrimateLit. Additionally, using the DSM-IV criteria and ethograms as guides, we developed behaviorally anchored alternative criteria that were applied to the case reports. A small number of chimpanzees in the case studies met DSM-IV criteria for PTSD and depression. Measures of inter-rater reliability, including Fleiss’ kappa and percentage agreement, were higher with use of the alternative criteria for PTSD and depression. In phase 2, the alternative criteria were applied to chimpanzees living in wild sites in Africa (n = 196) and chimpanzees living in sanctuaries with prior histories of experimentation, orphanage, illegal seizure, or violent human conflict (n = 168). In phase 2, 58% of chimpanzees living in sanctuaries met the set of alternative criteria for depression, compared with 3% of chimpanzees in the wild (p = 0.04), and 44% of chimpanzees in sanctuaries met the set of alternative criteria for PTSD, compared with 0.5% of chimpanzees in the wild (p = 0.04). Conclusions/Significance: Chimpanzees display behavioral clusters similar to PTSD and depression in their key diagnostic criteria, underscoring the importance of ethical considerations regarding the use of chimpanzees in experimentation and other captive settings

Signs of mood and anxiety disorders in chimpanzees.

BACKGROUND: In humans, traumatic experiences are sometimes followed by psychiatric disorders. In chimpanzees, studies have demonstrated an association between traumatic events and the emergence of behavioral disturbances resembling posttraumatic stress disorder (PTSD) and depression. We addressed the following central question: Do chimpanzees develop posttraumatic symptoms, in the form of abnormal behaviors, which cluster into syndromes similar to those described in human mood and anxiety disorders? METHODOLOGY/PRINCIPAL FINDINGS: In phase 1 of this study, we accessed case reports of chimpanzees who had been reportedly subjected to traumatic events, such as maternal separation, social isolation, experimentation, or similar experiences. We applied and tested DSM-IV criteria for PTSD and major depression to published case reports of 20 chimpanzees identified through PrimateLit. Additionally, using the DSM-IV criteria and ethograms as guides, we developed behaviorally anchored alternative criteria that were applied to the case reports. A small number of chimpanzees in the case studies met DSM-IV criteria for PTSD and depression. Measures of inter-rater reliability, including Fleiss' kappa and percentage agreement, were higher with use of the alternative criteria for PTSD and depression. In phase 2, the alternative criteria were applied to chimpanzees living in wild sites in Africa (n = 196) and chimpanzees living in sanctuaries with prior histories of experimentation, orphanage, illegal seizure, or violent human conflict (n = 168). In phase 2, 58% of chimpanzees living in sanctuaries met the set of alternative criteria for depression, compared with 3% of chimpanzees in the wild (p = 0.04), and 44% of chimpanzees in sanctuaries met the set of alternative criteria for PTSD, compared with 0.5% of chimpanzees in the wild (p = 0.04). CONCLUSIONS/SIGNIFICANCE: Chimpanzees display behavioral clusters similar to PTSD and depression in their key diagnostic criteria, underscoring the importance of ethical considerations regarding the use of chimpanzees in experimentation and other captive settings

Prevalence of psychiatric disorders among chimpanzees living in the wild and in sanctuaries (Phase 2).

<p>Prevalence of psychiatric disorders among chimpanzees living in the wild and in sanctuaries (Phase 2).</p

Percentage of chimpanzees displaying proposed individual alternative criteria for PTSD in Phase 2.

<p>Percentage of chimpanzees displaying proposed individual alternative criteria for PTSD in Phase 2.</p

Case studies used in phase 1, sex, citation(s), and general histories.

<p>Case studies used in phase 1, sex, citation(s), and general histories.</p

Number of cases in Phase 1 diagnosed with post-traumatic stress disorder (PTSD) and major depression (MDD) using two different sets of criteria.

<p>Number of cases in Phase 1 diagnosed with post-traumatic stress disorder (PTSD) and major depression (MDD) using two different sets of criteria.</p