A single-nucleotide polymorphism (SNP) multiplex system: the association of five SNPs with human eye and hair color in the Slovenian population and comparison using a Bayesian network and logistic regression model

Aim

To analyze two phenotype characteristics – eye and hair color – using single-nucleotide polymorphisms (SNPs) and evaluate their prediction accuracy in Slovenian population.

Methods

Twelve SNPs (OCA2 – rs1667394, rs7170989, rs1800407, rs7495174; HERC2 – rs1129038, rs12913832; MC1R – rs1805005, rs1805008; TYR – rs1393350; SLC45A2 – rs16891982, rs26722; SLC24A5 – rs1426654) were used for the development of a single multiplex assay. The single multiplex assay was based on SNaPshot chemistry and capillary electrophoresis. In order to evaluate the accuracy of the prediction of eye and hair color, we used the logistic regression model and the Bayesian network model, and compared the parameters of both.

Results

The new single multiplex assay displayed high levels of genotyping sensitivity with complete profiles generated from as little as 62 pg of DNA. Based on a prior evaluation of all SNPs in a single multiplex, we focused on the five most statistically significant in our population in order to investigate the predictive value. The two prediction models performed reliably without prior ancestry information, and revealed very good accuracy for both eye and hair color. Both models determined the highest predictive value for rs12913832 (P < 0.0001), while the other four SNPs (rs1393350, rs1800407, rs1805008, and rs7495174) showed additional association for color prediction.

Conclusion

We developed a sensitive and reliable single multiplex genotyping assay. More samples from different populations should be analyzed before this assay could be used as one of the supplemental tools in tracing unknown individuals in more complicated crime investigations.Height, face structure, pigmentation of the eye, hair, and skin, the presence of freckles, and male baldness make up human externally visible characteristics (EVC). To be able to predict eye and hair color based solely on biological material left behind at a crime scene or obtained from dismembered missing persons, or even of disaster victims, is one of the major expectations from the routine forensic work in the near future (1). However, genetic understanding of human appearance is still in its infancy, mainly due to the fact that all EVCs are polygenic traits. This means that yields from a large number of different genes and the expression of these genes are further influenced by mutual interactions and environmental interactions (2). Above all, molecular mechanisms and functional protein assays must also be considered in order to really understand how allelic variation in pigmentation genes could result in such a diversity of phenotypes in different human populations (3). The human eye (iris) and hair color are one of the most highly polymorphic phenotypes in people of European origin. The non-brown iris colors and red hair are generally features of European origin resulting from positive selection in early European history. There are several hypotheses for positive selection that mainly occurred in the Baltic region and Northern Europe. These are most likely: UV exposure causing skin cancer, vitamin D deficiency, and even sexual selection (4,5). Most EVCs are complex traits with many genes and single nucleotide polymorphism (SNP) variations, so the right combination of SNPs is crucial for the correct prediction of eye and hair color. Several genome-wide association studies (GWAS) for pigmentation have revealed that SNPs within the HERC2, OCA2, MC1R, SLC24A5, SLC45A2, TYR, and ASIP (4,6-16) genes were most strongly associated with eye and hair color in European populations. The latest data have shown that the main iris color variation is associated with a highly evolutionarily preserved region in the HERC2 gene or within the short sequence between the HERC2 and OCA2 genes. It is assumed that these regions represent a regulatory region controlling the constitutive expression of OCA2 (4,11,12). As for iris color, it has also been explained that red hair color is mainly associated with polymorphisms in the MC1R gene (13,17). On the other hand, the variations of genes such as SLC24A5, SLC45A, HERC2, and ASIP seem to be responsible for influencing the shades of hair color from blond to black (18,19).In order to correctly predict human eye and hair color from genetic data for the Slovenian population, we compared two alternative prediction models that are nowadays used most often in this field of forensics – the Bayesian network model and the logistic regression model. These models were developed and compared on the basis of the informative SNPs selected from our single multiplex assay.     

How health care professionals confront and solve ethical dilemmas – a tale of two countries: Slovenia and Croatia

AimTo assess the differences in the way how Slovenian and Croatian health care professionals (HCPs) confront ethical dilemmas and perceive the role of hospital ethics committees (HECs).MethodsThis cross-sectional, survey-based study involved HCPs from three Slovenian and five Croatian university medical centers (UMC). The final sample sizes were 308 (244 or 79.2% women) for Slovenia and 485 (398 or 82.1% women) for Croatia.ResultsCompared with Croatian physicians, Slovenian physicians reported a higher share of ethical dilemmas regarding waiting periods for diagnostics or treatment, suboptimal working conditions due to interpersonal relationships in the ward, and end-of-life treatment withdrawal, and a lower share regarding access to palliative care and patient information protection. Compared with Croatian nurses, Slovenian nurses reported a lower share of ethical dilemmas regarding the distribution of limited resources, recognizing the patient’s best interests, and access to palliative care. Compared with Croatian other HCPs, Slovenian other HCPs reported a lower burden of ethical dilemmas regarding waiting periods for diagnostics or treatment, distribution of limited resources, and access to palliative care. When encountering an ethical dilemma, all HCPs in both countries would first consult their colleagues. Slovenian and Croatian HCPs recognized the importance of the HECs to a similar extent, but viewed their role differently.ConclusionCroatian and Slovenian HCPs are confronted with different ethical dilemmas and perceive the role of HECs differently.

An ethical dilemma arises when we are confronted with a situation with two morally justifiable solutions, none of which is entirely satisfactory (1). In the course of their daily work, health care professionals (HCPs) encounter a broad range of ethical dilemmas (2-4), which often result in a moral distress for HCPs (5,6). A critical requirement for a successful response to an ethical dilemma is a strong foundation in medical professionalism cultivated during medical training and consolidated during professional work experience and career development (7-9).Slovenia and Croatia, previously the westernmost republics of the former Yugoslavia and now European Union members, share the same historical, geopolitical, economic, and religious background. A recent survey in the largest Slovenian tertiary hospital, the University Medical Center Ljubljana, found that the most important contexts that give rise to ethical dilemmas among HCPs were waiting periods for diagnostics and treatment, suboptimal working conditions due to poor interpersonal relationships, and preserving patients'' dignity, while the least important contexts were biomedical research, organ transplantation, and vaccine hesitancy (10). A study at the University Medical Center Rijeka found similar main ethical dilemmas in Croatian nurses and physicians, which included limiting life-sustaining therapy, euthanasia, and physician-assisted suicide (11).Except these two studies, little to nothing is known about the ethical dilemmas of HCPs in Slovenia and Croatia. In response to this limited evidence, we conducted a prospective survey with a primary objective to assess the differences in the share of ethical dilemmas among different categories of HCPs (physicians, nurses, and other HCPs) in Slovenian and Croatian tertiary hospitals (university medical centers, UMCs). The UMCs were purposively selected because in this kind of hospitals, one encounters complicated cases usually referred from other health care institutions for complex diagnostic and therapeutic procedures, which can often raise ethical issues. The secondary objectives of our survey were to study differences in the opinion on the existence of standard procedures when HCPs are facing an ethical dilemma; to determine whom HCPs consult when facing an ethical dilemma; and to identify the opinion on the importance of hospital ethics committees (HECs) and their role in Slovenia and Croatia.     

Correction to: Development of a conceptual model and patient-reported outcome measures for assessing symptoms and functioning in patients with heart failure

Quality of Life Research - The article Development of a conceptual model and patient-reported outcome measures for assessing symptoms and functioning in patients with heart failure.     

Evaluating Matching‐Adjusted Indirect Comparisons in Practice: A Case Study of Patients with Attention‐Deficit/Hyperactivity Disorder

Differences in patient characteristics across trials may bias efficacy estimates from indirect treatment comparisons. To address this issue, matching‐adjusted indirect comparison (MAIC) measures treatment efficacy after weighting individual patient data to match patient characteristics across trials. To date, however, there is no consensus on how best to implement MAIC. To address this issue, we applied MAIC to measure how two attention‐deficit/hyperactivity disorder (ADHD) treatments (guanfacine extended release and atomoxetine hydrochloride) affect patients' ADHD symptoms, as measured by the ADHD Rating Scale IV score. We tested MAIC sensitivity to: matched patient characteristics, matched statistical moments, weighting matrix, and placebo‐arm matching (i.e., matching on outcomes in the placebo arm). After applying MAIC, guanfacine and atomoxetine had similar reductions in ADHD symptoms (Δ: 0.4, p < 0.737). The results were similar for three of four sensitivity analyses. When we applied MAIC with placebo‐arm matching, however, guanfacine reduced symptoms more than atomoxetine (Δ: ?3.9, p < 0.004). We discuss the implication of this finding and advise MAIC practitioners to carefully consider the use of placebo‐arm matching, depending on the presence of residual confounding across trials. Copyright © 2016 John Wiley & Sons, Ltd.     

Shear-wave sonoelastographic features of invasive lobular breast cancers

AimTo evaluate shear-wave elastographic (SWE) and related gray-scale features of pure invasive lobular breast carcinoma (ILC) and compare them with invasive ductal breast cancers (IDC).MethodsQuantitative SWE features of mean (El-mean), maximum (El-max), minimum (El-min) elasticity values of the stiffest portion of the mass, and lesion-to-fat elasticity ratio (E-ratio) were measured in 40 patients with pure ILC and compared with 75 patients with IDC. Qualitative gray-scale features of lesion size, echogenicity, orientation, and presence of distal shadowing were determined and compared between the groups.ResultsILC were significantly larger than IDC (P = 0.008) and exhibited significantly higher El-max (P = 0.015) and higher El-mean (P = 0.008) than IDC. ILC were significantly more often horizontally oriented, while IDC were significantly more often vertically oriented (P < 0.001); ILC were significantly more often hyperechoic than IDC (P < 0.001). Differences in stiffness between ILC and IDC determined by quantitative SWE parameters were present only in small tumors (≤1.5 cm in size), ie, small ILC had significantly higher El-max (P = 0.030), El-mean (P = 0.014), and El-min (P = 0.045) than small IDC, while tumors larger than 1.5 cm had almost equal stiffness, without significant differences between the groups.ConclusionSpecific histopathologic features of ILC are translated into their qualitative sonographic and quantitative sonoelastographic appearance, with higher stiffness of small ILC compared to small IDC. Gray-scale and sonoelastographic features may help in diagnosing ILC.Invasive ductal cancer (IDC) is the most common breast cancer, while invasive lobular cancer (ILC) is the second most common and accounts for 6%-12% of breast cancers (1-3). ILC differs considerably from IDC by having a unique pathological growth pattern, the so called Indian-file pattern, with sheets of single-cell layers growing along the Cooper ligaments, ductuli, and other breast structures, resembling a spiderweb that diffusely spreads in the breast, producing minor desmoplastic reaction (4,5). This spiderweb-like growth is reflected in imaging features of ILC, as well as in its clinical presentation (6). IDC usually clinically manifests as a firm lump, while ILC usually manifests as a palpable thickening and skin or nipple retraction (3,5). ILC has increased tendency for multifocality and multicentricity, a higher risk of bilateral breast cancer (20%-29%), and older age at onset (7,8). Lymph node metastases are less common in ILC than in IDC of equal size, because ILC tumor cells lack cellular atypia and often have low mitotic rate (9). ILC has the propensity to metastasize to the chest, peritoneum, retroperitoneum, and pelvis (10).Because of its growth pattern of mass infiltrating surrounding tissues, IDC is much more easily detected than ILC also on mammography. ILC has higher false-negative mammographic rates than IDC, since ILC may be invisible or may have quite low mammographic density, and microcalcifications are uncommon (6,11). Due to the higher propensity for multicentric and bilateral lesions, it is generally considered that patients with ILC should be referred to preoperative breast MRI, the best imaging modality to evaluate the tumor extent, while the benefit for preoperative MRI in IDC has not yet been proven (12,13). Fine-needle aspiration is not as sensitive for the diagnosis of ILC as it is for IDC, and core-biopsy should be performed when ILC is suspected, even in cases of palpable lesions (14,15). ILC is associated more often than IDC with positive margins on surgical excision and is more often treated with mastectomy, because of the large size at diagnosis and underestimation of tumor extent with conventional imaging (16).Ultrasound of the breast is widely used in the diagnosis of breast cancer, usually after mammography, and most image-guided core biopsies of breast lesions are routinely performed under the sonographic guidance (17,18). Ultrasound is highly operator-dependent, much more than mammography or MRI. The quality of ultrasonic equipment and transducers is variable, suboptimal examinations are common, and interobserver variability is high; sensitivity of ultrasound in detection of ILC is reported in the range of 68%-88% (6,12,19).Sonoelastography is a relatively new ultrasonographic method, which may help in the detection and differentiation of benign and malignant breast lesions (18,20). Strain elastography allows qualitative estimation of the breast lesion stiffness, while shear-wave elastography (SWE) allows quantification of lesion stiffness in kilopascals (kPa) (18). Multicentric studies found that SWE features can help discriminate breast cancers and benign breast lesions, and breast cancers among themselves (20-22). It was also shown that some IDC, like triple negative breast cancers, differ in their stiffness compared to other IDC (23). Studies evaluating some SWE features of invasive cancers were done in a small number of patients with ILC, but to the best of our knowledge none so far has provided values specific for a larger, homogeneous group of patients with pure ILC (24,25).The aim of this single-center study was to evaluate and establish SWE and related conventional sonographic features of pure ILC of the breast in a group of 40 patients, and to compare these features with the most common invasive breast cancer, IDC. SWE features within ILC group were also correlated with tumor size, extent, histologic grade, and the presence of nodal metastases.     

A panel of DNA methylation markers for the classification of consensus molecular subtypes 2 and 3 in patients with colorectal cancer

Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). We aim to identify methylation markers to distinguish between CMS2 and CMS3 in patients with CRC, for which an easy test is currently lacking. To this aim, fresh‐frozen tumor tissue of 239 patients with stage I‐III CRC was analyzed. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group‐regularized logistic ridge regression with post hoc group‐weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3. The Cancer Genome Atlas (TCGA) data were used for validation. Group regularization of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier, resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only five markers, accuracies were > 90% in our cohort and in the TCGA validation set. Our methylation marker panel accurately distinguishes between CMS2 and CMS3. This enables development of a targeted assay to provide a robust and clinically relevant classification tool for CRC patients.     

Synergistic Effect of Screen-Printed Single-Walled Carbon Nanotubes and Phosphorylated Cellulose Nanofibrils on Thermophysiological Comfort,Thermal/UV Resistance,Mechanical and Electroconductive Properties of Flame-Retardant Fabric

Single-walled carbon nanotubes (SWCNTs) and phosphorylated nanocellulose fibrils (PCNFs) were used as functional screen-print coatings on flame-retardant (FR) fabric, to improve its thermal resistance and thermophysiological comfort (wetting, water vapour and heat transmission) properties, while inducing it with electrical conductivity and UV protection. The effect of PCNF printing, followed by applying a hydrophobic polyacrylate (AP), on the same (back/B, turned outwards) or other (front/F, turned towards skin) side of the fabric, with and without the addition of 0.1–0.4 wt% SWCNTs, was studied by determining the amount of applied coating and its distribution (microscopic imaging), and measuring the fabric’s colour, air permeability, thickness, mechanical, flame and abrasion resistance properties. Due to the synergistic effect of PCNF and SWCNTs, both-sided printed fabric (front-side printed with PCNF and back-side with SWCNTs within AP) resulted in an increased heat transfer (25%) and an improved thermal resistance (shift of degradation temperature by up to 18 °C towards a higher value) and UV protection (UPF of 109) without changing the colour of the fabric. Such treatment also affected the moisture management properties with an increased water-vapour transfer (17%), reduced water uptake (39%) and asymmetric wettability due to the hydrophilic front (Contact Angle 46°) and hydrophobic back (129°) side. The increased tensile (16%) and tear (39%) strengths were also assessed in the warp direction, without worsening the abrasion resistance of the front-side. A pressure-sensing electrical conductivity (up to 4.9∙10⁻⁴ S/cm with an increase to 12.0∙10⁻⁴ S/cm at 2 bars) of the SWCNT-printed side ranks the fabric among the antistatic, electrostatic discharge (ESD) or electromagnetic interference (EMI) shielding protectives.     

Gene expression profiling assigns CHEK2 1100delC breast cancers to the luminal intrinsic subtypes

CHEK2 1100delC is a moderate-risk cancer susceptibility allele that confers a high breast cancer risk in a polygenic setting. Gene expression profiling of CHEK2 1100delC breast cancers may reveal clues to the nature of the polygenic CHEK2 model and its genes involved. Here, we report global gene expression profiles of a cohort of 155 familial breast cancers, including 26 CHEK2 1100delC mutant tumors. In line with previous work, all CHEK2 1100delC mutant tumors clustered among the hormone receptor-positive breast cancers. In the hormone receptor-positive subset, a 40-gene CHEK2 signature was subsequently defined that significantly associated with CHEK2 1100delC breast cancers. The identification of a CHEK2 gene signature implies an unexpected biological homogeneity among the CHEK2 1100delC breast cancers. In addition, all 26 CHEK2 1100delC tumors classified as luminal intrinsic subtype breast cancers, with 8 luminal A and 18 luminal B tumors. This biological make-up of CHEK2 1100delC breast cancers suggests that a relatively limited number of additional susceptibility alleles are involved in the polygenic CHEK2 model. Identification of these as-yet-unknown susceptibility alleles should be aided by clues from the 40-gene CHEK2 signature.