In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

Background: There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (~23.8 Gb/C). [br/] Methodology/Principal Findings: A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). [br/] Conclusions/Significance: This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome

Rare variants in the CYP27B1 gene are associated with multiple sclerosis.

OBJECTIVE: Multiple sclerosis (MS) is a complex neurological disease. Genetic linkage analysis and genotyping of candidate genes in families with 4 or more affected individuals more heavily loaded for susceptibility genes has not fully explained familial disease clustering. METHODS: We performed whole exome sequencing to further understand the heightened prevalence of MS in these families. RESULTS: Forty-three individuals with MS (1 from each family) were sequenced to find rare variants in candidate MS susceptibility genes. On average, >58,000 variants were identified in each individual. A rare variant in the CYP27B1 gene causing complete loss of gene function was identified in 1 individual. Homozygosity for this mutation results in vitamin D-dependent rickets I (VDDR1), whereas heterozygosity results in lower calcitriol levels. This variant showed significant heterozygous association in 3,046 parent-affected child trios (p = 1 × 10(-5)). Further genotyping in >12,500 individuals showed that other rare loss of function CYP27B1 variants also conferred significant risk of MS, Peto odds ratio = 4.7 (95% confidence interval, 2.3-9.4; p = 5 × 10(-7)). Four known VDDR1 mutations were identified, all overtransmitted. Heterozygous parents transmitted these alleles to MS offspring 35 of 35× (p = 3 × 10(-9)). INTERPRETATION: A causative role for CYP27B1 in MS is supported; the mutations identified are known to alter function having been shown in vivo to result in rickets when 2 copies are present. CYP27B1 encodes the vitamin D-activating 1-alpha hydroxylase enzyme, and thus a role for vitamin D in MS pathogenesis is strongly implicated

An AP4B1 frameshift mutation in siblings with intellectual disability and spastic tetraplegia further delineates the AP-4 deficiency syndrome

Abdollahpour H, Alawi M, Kortüm F, et al. An AP4B1 frameshift mutation in siblings with intellectual disability and spastic tetraplegia further delineates the AP-4 deficiency syndrome. European Journal of Human Genetics. 2015;23(2):256-259.The recently proposed adaptor protein 4 (AP-4) deficiency syndrome comprises a group of congenital neurological disorders characterized by severe intellectual disability (ID), delayed or absent speech, hereditary spastic paraplegia, and growth retardation. AP-4 is a heterotetrameric protein complex with important functions in vesicle trafficking. Mutations in genes affecting different subunits of AP-4, including AP4B1, AP4E1, AP4S1, and AP4M1, have been reported in patients with the AP-4 deficiency phenotype. We describe two siblings from a non-consanguineous couple who presented with severe ID, absent speech, microcephaly, growth retardation, and progressive spastic tetraplegia. Whole-exome sequencing in the two patients identified the novel homozygous 2-bp deletion c.1160_1161delCA (p.(Thr387Argfs*30)) in AP4B1. Sanger sequencing confirmed the mutation in the siblings and revealed it in the heterozygous state in both parents. The AP4B1-associated phenotype has previously been assigned to spastic paraplegia-47. Identification of a novel AP4B1 alteration in two patients with clinical manifestations highly similar to other individuals with mutations affecting one of the four AP-4 subunits further supports the observation that loss of AP-4 assembly or functionality underlies the common clinical features in these patients and underscores the existence of the clinically recognizable AP-4 deficiency syndrome

Entrepreneurial resilience

The final publication is available at Springer via DOI TBCThe vast majority of businesses in all countries - between 70% and 95% - are micro-businesses, i.e. enterprises that employ fewer than ten people (OECD, 2017). Their impact on the economies and societies in which they operate is therefore significant, collectively acting as important sources of employment, growth and innovation (ibid, 2017). However, the existence of many of these businesses is often precarious, especially in the early stages of their development. Many newly created businesses fail within the first few years of life with mortality rates ranging from around 10% (UK, USA, Sweden) to 45% (Slovak Republic) in the first year (ibid, 2017). As a result, the entrepreneurial activity to create and manage these businesses is very demanding and exposes entrepreneurs to situations which would be expected to create high levels of stress among the general population (e.g. a rapidly changing and unpredictable environment, high responsibility, high workload). The demands of business start-up and ownership could be expected to create a higher risk of mental health problems. Isolation and long working hours could contribute to an increased risk of depression. Moreover, for many entrepreneurs, their business ventures are personal passions and their self-worth and well-being can be intimately connected to the success of those ventures (Murnieks, Mosakowski and Cardon, 2014). On a practical level, the pressures are often high and can create anxiety as personal financial well-being is often directly related to the ability to close the next deal. Furthermore, Spivak, McKelvie and Haynie (2014) highlight a possible “dark side” of entrepreneurship outcomes, finding that habitual entrepreneurs can suffer from symptoms of behavioural addictions - withdrawal-engagement patterns, obsessive thoughts, and negative emotions - arising from repeated venture creation activities. However, at the same time, Baron, Franklin and Hmieleski (2016) find that entrepreneurs experience lower stress compared to other occupational groups when creating new ventures. Baron et al (2016) suggest self-selection effects as the underlying mechanism producing entrepreneurs that are above average (as a group) in their capacity to handle stress effectively, arguing that those who persist in entrepreneurship acquire this capacity, the resilience to handle the stressors and challenges of their entrepreneurial context