Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss

Inactivating variants as well as a missense variant in the centrosomal CEP78 gene have been identified in autosomal recessive cone-rod dystrophy with hearing loss (CRDHL), a rare syndromic inherited retinal disease distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the CEP78 region and characterization of their underlying mechanisms. Approaches used for the identification of the SVs are shallow whole-genome sequencing (sWGS) combined with quantitative polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) data. Targeted or whole-genome nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SVs cases, the effect of the SVs on CEP78 expression was assessed using quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), two SVs affecting CEP78 were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235 kb and a partial gene deletion of 15 kb in a heterozygous and homozygous state, respectively. Assessment of the molecular consequences of the SVs on patient’s materials displayed a loss-of-function effect. Delineation and characterization of the 15-kb deletion using targeted LRS revealed the previously described complex CEP78 SV, suggestive of a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in cases of Belgian and British origin, respectively. The novel 235-kb deletion was delineated using whole-genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based underlying mechanism. Moreover, data mining of bulk and single-cell human and mouse transcriptional datasets, together with CEP78 immunostaining on human retina, linked the CEP78 expression domain with its phenotypic manifestations. Overall, this study supports that the CEP78 locus is prone to distinct SVs and that SV analysis should be considered in a genetic workup of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole-genome LRS in identifying and characterizing complex SVs in patients with ocular diseases

The principle of respect for autonomy – Concordant with the experience of oncology physicians and molecular biologists in their daily work?

<p>Abstract</p> <p>Background</p> <p>This article presents results from a qualitative empirical investigation of how Danish oncology physicians and Danish molecular biologists experience the principle of respect for autonomy in their daily work.</p> <p>Methods</p> <p>This study is based on 12 semi-structured interviews with three groups of respondents: a group of oncology physicians working in a clinic at a public hospital and two groups of molecular biologists conducting basic research, one group employed at a public university and the other in a private biopharmaceutical company.</p> <p>Results</p> <p>We found that that molecular biologists consider the principle of respect for autonomy as a negative obligation, where the informed consent of patients or research subjects should be respected. Furthermore, molecular biologists believe that very sick patients are constraint by the circumstances to a certain choice. However, in contrast to molecular biologists, oncology physicians experience the principle of respect for autonomy as a positive obligation, where the physician in dialogue with the patient performs a medical prognosis based on the patient's wishes and ideas, mutual understanding and respect. Oncology physicians believe that they have a positive obligation to adjust to the level of the patient when providing information making sure that the patient understands. Oncology physicians experience situations where the principle of respect for autonomy does not apply because the patient is in a difficult situation.</p> <p>Conclusion</p> <p>In this study we explore the moral views and attitudes of oncology physicians and molecular biologists and compare these views with bioethical theories of the American bioethicists Tom L. Beauchamp & James F. Childress and the Danish philosophers Jakob Rendtorff & Peter Kemp. This study shows that essential parts of the two bioethical theories are reflected in the daily work of Danish oncology physicians and Danish molecular biologists. However, the study also explores dimensions where the theories can be developed further to be concordant with biomedical practice. The hope is that this study enhances the understanding of the principle of respect for autonomy and the way it is practiced.</p

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments

Follicle Stimulating Hormone is an accurate predictor of azoospermia in childhood cancer survivors

Funding: RTM is supported by a Wellcome Trust Intermediate Clinical Fellowship (grant no: 098522), https://wellcome.ac.uk/what-we-do/directories/intermediate-clinical-fellowships-people-funded. TWK is supported by Engineering and Physical Sciences Research Council grant EP/P015638/1, http://gow.epsrc.ac.uk/NGBOViewGrant.aspx?GrantRef=EP/P015638/1.The accuracy of Follicle Stimulating Hormone as a predictor of azoospermia in adult survivors of childhood cancer is unclear, with conflicting results in the published literature. A systematic review and post hoc analysis of combined data (n = 367) were performed on all published studies containing extractable data on both serum Follicle Stimulating Hormone concentration and semen concentration in survivors of childhood cancer. PubMed and Medline databases were searched up to March 2017 by two blind investigators. Articles were included if they contained both serum FSH concentration and semen concentration, used World Health Organisation certified methods for semen analysis, and the study participants were all childhood cancer survivors. There was no evidence for either publication bias or heterogeneity for the five studies. For the combined data (n = 367) the optimal Follicle Stimulating Hormone threshold was 10.4 IU/L with specificity 81% (95% CI 76%–86%) and sensitivity 83% (95% CI 76%–89%). The AUC was 0.89 (95%CI 0.86–0.93). A range of threshold FSH values for the diagnosis of azoospermia with their associated sensitivities and specificities were calculated. This study provides strong supporting evidence for the use of serum Follicle Stimulating Hormone as a surrogate biomarker for azoospermia in adult males who have been treated for childhood cancer.Publisher PDFPeer reviewe

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.</p

Severity of Giardia infection associated with post-infectious fatigue and abdominal symptoms two years after

<p>Abstract</p> <p>Background</p> <p>A high rate of post-infectious fatigue and abdominal symptoms two years after a waterborne outbreak of giardiasis in Bergen, Norway in 2004 has previously been reported. The aim of this report was to identify risk factors associated with such manifestations.</p> <p>Methods</p> <p>All laboratory confirmed cases of giardiasis (n = 1262) during the outbreak in Bergen in 2004 received a postal questionnaire two years after. Degree of post-infectious abdominal symptoms and fatigue, as well as previous abdominal problems, was recorded. In the statistical analyses number of treatment courses, treatment refractory infection, delayed education and sick leave were used as indices of protracted and severe <it>Giardia </it>infection. Age, gender, previous abdominal problems and symptoms during infection were also analysed as possible risk factors. Simple and multiple ordinal logistic regression models were used for the analyses.</p> <p>Results</p> <p>The response rate was 81% (1017/1262), 64% were women and median age was 31 years (range 3-93), compared to 61% women and 30 years (range 2-93) among all 1262 cases. Factors in multiple regression analysis significantly associated with abdominal symptoms two years after infection were: More than one treatment course, treatment refractory infection, delayed education, bloating and female gender. Abdominal problems prior to <it>Giardia </it>infection were not associated with post-infectious abdominal symptoms. More than one treatment course, delayed education, sick leave more than 2 weeks, and malaise at the time of infection, were significantly associated with fatigue in the multiple regression analysis, as were increasing age and previous abdominal problems.</p> <p>Conclusion</p> <p>Protracted and severe <it>giardiasis </it>seemed to be a risk factor for post-infectious fatigue and abdominal symptoms two years after clearing the <it>Giardia </it>infection.</p

The Meaning of Body Experience Evaluation in Oncology

Evaluation of quality of life, psychic and bodily well-being is becoming increasingly important in oncology aftercare. This type of assessment is mainly carried out by medical psychologists. In this paper I will seek to show that body experience valuation has, besides its psychological usefulness, a normative and practical dimension. Body experience evaluation aims at establishing the way a person experiences and appreciates his or her physical appearance, intactness and competence. This valuation constitutes one’s ‘body image’. While, first, interpreting the meaning of body image and, second, indicating the limitations of current psychological body image assessment, I argue that the normative aspect of body image is related to the experience of bodily wholeness or bodily integrity. Since this experience is contextualized by a person’s life story, evaluation should also focus on narrative aspects. I finally suggest that the interpretation of body experience is not only valuable to assess a person’s quality of life after treatment, but that it can also be useful in counseling prior to interventions, since it can support patients in making decisions about interventions that will change their bodies. To apply this type of evaluation to oncology practice, a rich and tailored vocabulary of body experiences has to be developed

Not gods but animals : human dignity and vulnerable subjecthood

Drawing on earlier work on the conceptual structure of dignity, this paper will suggest a particular type of connectedness between vulnerability and human dignity; namely, that the ‘‘organizing idea’’ of human dignity is the idea of a particular sort of ethical response to universal human vulnerability. It is common ground among many, if not all, approaches to ethics that vulnerability requires us to respond ethically. Here, I argue that human dignity is distinctive among ethical values in that it values us because of, rather than in spite of, or regardless of, our universal vulnerability. The term ‘‘dignity’’ is used synonymously with ‘‘human dignity’’ here, since an investigation of the dignity of non-human entities forms no part of the present examination