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

A Multi-Organ-on-Chip Approach to Investigate How Oral Exposure to Metals Can Cause Systemic Toxicity Leading to Langerhans Cell Activation in Skin

Investigating systemic toxicity in vitro is still a huge challenge. Here, a multi-organ-on-chip approach is presented as a typical case of topical exposure of oral mucosa to metals, which are known to activate the immune system and in turn may result in skin inflammation. Reconstructed human gingiva (RHG) and reconstructed human skin containing MUTZ-3-derived Langerhans cells (MUTZ-LC) in the epidermis (RHS-LC) were incorporated into a HUMIMIC Chip3plus, connected by dynamic flow and cultured for a total period of 72 h. Three independent experiments were performed each with an intra-experiment replicate in order to assess the donor and technical variations. After an initial culture period of 24 h to achieve stable dynamic culture conditions, nickel sulfate was applied topically to RHG for 24 h, and LC activation (maturation and migration) was determined in RHS-LC after an additional 24 h incubation time. A stable dynamic culture of RHG and RHS-LC was achieved as indicated by the assessment of glucose uptake, lactate production, and lactate dehydrogenase release into the microfluidics compartment. Nickel exposure resulted in no major histological changes within RHG or RHS-LC, or cytokine release into the microfluidics compartment, but did result in an increased activation of LC as observed by the increased mRNA levels of CD1a, CD207, HLA-DR, and CD86 in the dermal compartment (hydrogel of RHS-LC (PCR)). This is the first study to describe systemic toxicity and immune cell activation in a multi-organ setting and can provide a framework for studying other organoids in the future

Exploring DNA methylation patterns in copper exposed Folsomia candida and Enchytraeus crypticus

Accumulating evidence shows that epigenetics-mediated phenotypic plasticity plays a role in an organism's ability to deal with environmental stress. However, to date, the role of epigenetic modifications in response to stress is hardly investigated in soil invertebrates. The main objective of this proof of principle study was to explore whether total cytosine and locus-specific CpG methylation are present in two important ecotoxicological model organisms, the springtail Folsomia candida and the potworm Enchytraeus crypticus, and if so, whether methylation patterns might change with increased toxicant exposure. LC–MS/MS analyses and bisulfite sequencing were performed to identify the CpG methylation state of the organisms. We show here, for the first time, a total level of 1.4% 5-methyl cytosine methylation in the genome of E. crypticus, and an absence of both total cytosine and locus-specific CpG methylation in F. candida. In E. crypticus, methylation of CpG sites was observed in the coding sequence (CDS) of the housekeeping gene Elongation Factor 1α while the CDS of the stress inducible Heat Shock Protein 70 gene almost lacked methylation. This confirms previous observations that DNA methylation differs between housekeeping and stress-inducible genes in invertebrates. DNA methylation patterns in E. crypticus were not affected by exposure to copper (II) sulfate pentahydrate (CuSO 4 ·5H 2 O) mixed in with LUFA 2.2 soil at sublethal effect concentrations that decreased reproduction by 10%, 20% and 50%. Although, differences in CpG methylation patterns between specific loci suggest a functional role for DNA methylation in E. crypticus, genome-wide bisulfite sequencing is needed to verify whether environmental stress affects this epigenetic hallmark

Exploring DNA methylation patterns in copper exposed Folsomia candida and Enchytraeus crypticus

Accumulating evidence shows that epigenetics-mediated phenotypic plasticity plays a role in an organism's ability to deal with environmental stress. However, to date, the role of epigenetic modifications in response to stress is hardly investigated in soil invertebrates. The main objective of this proof of principle study was to explore whether total cytosine and locus-specific CpG methylation are present in two important ecotoxicological model organisms, the springtail Folsomia candida and the potworm Enchytraeus crypticus, and if so, whether methylation patterns might change with increased toxicant exposure. LC–MS/MS analyses and bisulfite sequencing were performed to identify the CpG methylation state of the organisms. We show here, for the first time, a total level of 1.4% 5-methyl cytosine methylation in the genome of E. crypticus, and an absence of both total cytosine and locus-specific CpG methylation in F. candida. In E. crypticus, methylation of CpG sites was observed in the coding sequence (CDS) of the housekeeping gene Elongation Factor 1α while the CDS of the stress inducible Heat Shock Protein 70 gene almost lacked methylation. This confirms previous observations that DNA methylation differs between housekeeping and stress-inducible genes in invertebrates. DNA methylation patterns in E. crypticus were not affected by exposure to copper (II) sulfate pentahydrate (CuSO 4 ·5H 2 O) mixed in with LUFA 2.2 soil at sublethal effect concentrations that decreased reproduction by 10%, 20% and 50%. Although, differences in CpG methylation patterns between specific loci suggest a functional role for DNA methylation in E. crypticus, genome-wide bisulfite sequencing is needed to verify whether environmental stress affects this epigenetic hallmark

A regulatory CD4+ T cell subset in the BB rat model of autoimmune diabetes expresses neither CD25 nor Foxp3

Biobreeding (BB) rats model type 1 autoimmune diabetes (T1D). BB diabetes-prone (BBDP) rats develop T1D spontaneously. BB diabetes-resistant (BBDR) rats develop T1D after immunological perturbations that include regulatory T cell (Treg) depletion plus administration of low doses of a TLR ligand, polyinosinic-polycytidylic acid. Using both models, we analyzed CD4+CD25+ and CD4+CD45RC- candidate rat Treg populations. In BBDR and control Wistar Furth rats, CD25+ T cells comprised 5-8% of CD4+ T cells. In vitro, rat CD4+CD25+ T cells were hyporesponsive and suppressed T cell proliferation in the absence of TGF-beta and IL-10, suggesting that they are natural Tregs. In contrast, CD4+CD45RC(-) T cells proliferated in vitro in response to mitogen and were not suppressive. Adoptive transfer of purified CD4+CD25+ BBDR T cells to prediabetic BBDP rats prevented diabetes in 80% of recipients. Surprisingly, CD4+CD45RC-CD25- T cells were equally protective. Quantitative studies in an adoptive cotransfer model confirmed the protective capability of both cell populations, but the latter was less potent on a per cell basis. The disease-suppressing CD4+CD45RC-CD25- population expressed PD-1 but not Foxp3, which was confined to CD4+CD25+ cells. We conclude that CD4+CD25+ cells in the BBDR rat act in vitro and in vivo as natural Tregs. In addition, another population that is CD4+CD45RC-CD25- also participates in the regulation of autoimmune diabetes

Neonatal screening for profound biotinidase deficiency in the Netherlands:consequences and considerations

Biotinidase deficiency is a rare inherited metabolic disorder that can cause severe neurological symptoms. To prevent severe clinical presentations, it was included in the Dutch neonatal screening programme in 2007. Since then the number of cases detected has been high. This study set out to describe the incidence of the disease, the clinical and demographic characteristics of the neonates identified and the type of mutations found. In the south-western Netherlands, 304 982 neonates were screened between 2007 and 2012; and 92 were identified for further testing. Confirmatory testing revealed 6 (7%) with a profound biotinidase deficiency (o10% enzyme activity), 44 (48%) with a partial deficiency (10-30%) and 42 (46%) with normal activity (> 30%). All six patients whose profound deficiency was confirmed had enzyme activities below 15% on neonatal screening. Mutation analysis was performed in 61 neonates: 5 'profound', 35 'partial' and 21 'normal'. All five 'profound' cases had two severe mutations. Comparison with the northern Netherlands showed that the frequency and types of mutation were representative for the Netherlands as a whole. The most common mutation detected was c.[1330G > C] (p.(Asp444His); 34%), which is considered to be mild, followed by three severe mutations c.[1368A > C], c.[1595C > T] and c.[1330G > C; 511G > A]. Seven new mutations were identified. We conclude that neonatal screening for profound biotinidase produces a high number of false positives. Biotinidase deficiency was profound in less than 10% of cases identified. As biotinidase activity lay below 15% on neonatal screening in all such cases, the screening threshold might be reduced to 15%