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The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at −40 °C prior to distribution and the participants are instructed to store the samples frozen at −20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25-hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3). A set of 50 single-donor samples was assigned target values for concentrations of 25(OH)D2, 25(OH)D3, 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3), and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) using isotope dilution liquid chromatography - tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 1 includes results from 14 laboratories using 14 custom LC-MS/MS assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 53% of the LC-MS/MS assays met the VDSP criterion of mean % bias ≤ |±5%|. For the LC-MS/MS assays not meeting the ≤ |±5%| criterion, four assays had mean % bias of between 12 and 21%. Based on multivariable regression analysis using the concentrations of the four individual vitamin D metabolites in the 50 single-donor samples, the performance of several LC-MS/MS assays was found to be influenced by the presence of 3-epi-25(OH)D3. The results of this interlaboratory study represent the most comprehensive comparison of LC-MS/MS assay performance for serum total 25(OH)D and document the significant impact of the lack of separation of 3-epi-25(OH)D3 and 25(OH)D3 on assay performance, particularly with regard to mean % bias.

An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials(®) (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography – tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D(2) [25(OH)D(2)] and 25-hydroxyvitamin D(3) [25(OH)D(3)] using Reference Measurement Procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D [i.e., the sum of 25(OH)D(2) and 25(OH)D(3)] determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D(2) below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D(2) were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (Level 4), which has high exogenous concentration of 3-epi-25(OH)D(3) was deemed non-commutable for 50% of the LC-MS/MS assays.

Background Respiratory failure and thromboembolism are frequent in severe acute respiratory syndrome coronavirus 2–infected patients. Vitamin K activates both hepatic coagulation factors and extrahepatic endothelial anticoagulant protein S, required for thrombosis prevention. In times of vitamin K insufficiency, hepatic procoagulant factors are preferentially activated over extrahepatic proteins. Vitamin K also activates matrix Gla protein (MGP), which protects against pulmonary and vascular elastic fiber damage. We hypothesized that vitamin K may be implicated in coronavirus disease 2019 (COVID-19), linking pulmonary and thromboembolic disease. Methods A total of 135 hospitalized COVID-19 patients were compared with 184 historic controls. Inactive vitamin K–dependent MGP (desphospho-uncarboxylated [dp-uc] MGP) and prothrombin (PIVKA-II) were measured inversely related to extrahepatic and hepatic vitamin K status, respectively. Desmosine was measured to quantify the rate of elastic fiber degradation. Arterial calcification severity was assessed using computed tomography. Results dp-ucMGP was elevated in COVID-19 patients compared with controls (P Conclusions dp-ucMGP was severely increased in COVID-19 patients, indicating extrahepatic vitamin K insufficiency, which was related to poor outcome; hepatic procoagulant factor II remained unaffected. These data suggest pneumonia-induced extrahepatic vitamin K depletion leading to accelerated elastic fiber damage and thrombosis in severe COVID-19 due to impaired activation of MGP and endothelial protein S, respectively.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.

Despite the increasing awareness about biotin interference with immunoassays, so far, only two studies have quantified the prevalence of elevated biotin in patient populations. In a US study, over 7% had biotin concentrations exceeding 10 ng/mL, whereas in an Australian study only 0.8% of ED samples contained biotin exceeding 10 ng/mL. At present, representative data for the European population are lacking. In this study, we investigated biotin prevalence in The Netherlands in a representative cohort of routine laboratory requests in our laboratory using an LC-MS/MS assay for quantification of biotin in human plasma. In our study, we found 0.2% of samples exceeding 10 ng/mL of biotin, a finding more or less in line with the Australian data. Even though the biotin prevalence appears to be low, with concomitant low to moderate biotin concentrations, it is by no means a rare phenomenon. Laboratories like ours are likely to experience biotin positive samples on a daily basis with variable impact on patient care depending on the analytical bias from the immunoassay platform used. Our simple and robust LC–MS/MS assay for quantification of biotin in human samples may contribute to better understanding of the systemic concentrations seen after moderate- and high-dose biotin supplementation and the extent of immunoassay interference.

We report a case of a 22-year-old female patient who was diagnosed with a cribriform-morular variant of papillary thyroid carcinoma (CMV-PTC). While at early ages this thyroid cancer variant is highly suggestive for familial adenomatous polyposis (FAP), there was no family history of FAP. In the tumor biallelic, inactivating APC variants were identified. The patient tested negative for germline variants based on analysis of genomic DNA from peripheral blood leukocytes. Somatic mosaicism was excluded by subsequent deep sequencing of leukocyte and normal thyroid DNA using next generation sequencing (NGS). This report presents a rare sporadic case of CMV-PTC, and to the best of our knowledge the first featuring two somatic APC mutations underlying the disease, with an overview of CMV-PTC cases with detected APC and CTNNB1 pathogenic variants from the literature. Electronic supplementary material The online version of this article (10.1007/s10689-019-00146-4) contains supplementary material, which is available to authorized users.

BackgroundPathology during COVID-19 infection arises partly from an excessive inflammatory response with a key role for interleukin (IL)-6. Both vitamin D and K have been proposed as potential modulators of this process.MethodsWe assessed vitamin D and K status by measuring circulating 25-hydroxyvitamin D (25(OH)D) and desphospho-uncarboxylated Matrix Gla-Protein (dp-ucMGP), respectively in 135 hospitalized COVID-19 patients in relation to inflammatory response, elastic fiber degradation and clinical outcomes.ResultsComparing good and poor disease outcomes of COVID-19 patients, vitamin 25(OH)D levels were not significantly different. IL-6 levels, however, were significantly higher in patients with poor outcome, compared to patients with good outcome (30.3 vs. 153.0 pg/mL; p < 0.0001). Dp-ucMGP levels as biomarker of extrahepatic vitamin K status was associated with IL-6 levels (r = 0.35; p < 0.0001). In contrast, 25(OH)D levels were only borderline statistically significant correlated with IL-6 (r = −0.14; p ConclusionsDp-ucMGP associates with IL-6 as a central component of the destructive inflammatory processes in COVID-19. An intervention trial may provide insight whether vitamin K administration, either or not in combination with vitamin D, improves clinical outcome of COVID-19.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25-hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D

Vitamin D is a nutrient with anti-inflammatory properties whose role is currently being evaluated in COVID-19. Although studies are conflicting, they seem to suggest a role for vitamin D in reducing disease susceptibility but not in improving clinical outcome. In order to understand why vitamin D does not seem to have much effect on decreasing disease severity, it is essential to appreciate pulmonary vitamin D metabolism. To reach the pulmonary compartment, vascular endothelial cells would need to take up vitamin D from the blood stream, but they lack vitamin D receptor (VDR) and the activating enzyme CYP27B1. Endothelialitis – an important disease manifestation of COVID-19 – is therefore not expected to be directly affected by vitamin D. Bronchial epithelial cells are usually among the first to be infected with SARS-CoV-2. They do express both VDR and CYP27B1, but circulating vitamin D may not reach bronchial epithelial cells without transportation from the blood stream through the blood vessel wall. Inhalation therapy with vitamin D has therefore been suggested as an alternative for oral administration to bypass endothelial cells and efficaciously target bronchial epithelium. In conclusion, based on the principles of pulmonary vitamin D metabolism, it is not expected that vitamin D administration has a significant effect on COVID-19 severity. Vitamin D is more likely to reduce SARS-CoV-2 susceptibility, but reaching the airways with oral supplementation will be difficult and vitamin D inhalation therapy should be considered.

Background Vitamin D supplementation lowers exacerbation frequency in severe vitamin D-deficient patients with COPD. Data regarding the effect of vitamin D on elastin degradation are lacking. Based on the vitamin's anti-inflammatory properties, we hypothesised that vitamin D supplementation reduces elastin degradation, particularly in vitamin D-deficient COPD patients. We assessed the effect of vitamin D status and supplementation on elastin degradation by measuring plasma desmosine, a biomarker of elastin degradation. Methods Desmosine was measured every 4 months in plasma of 142 vitamin D-naïve COPD patients from the Leuven vitamin D intervention trial (100 000 IU vitamin D3 supplementation every 4 weeks for 1 year). Results No significant association was found between baseline 25-hydroxyvitamin D (25(OH)D) and desmosine levels. No significant difference in desmosine change over time was found between the placebo and intervention group during the course of the trial. In the intervention arm, an unexpected inverse association was found between desmosine change and baseline 25(OH)D levels (p=0.005). Conclusions Vitamin D supplementation did not have a significant overall effect on elastin degradation compared to placebo. Contrary to our hypothesis, the intervention decelerated elastin degradation in vitamin D-sufficient COPD patients and not in vitamin D-deficient subjects., Vitamin D supplementation does not have a significant overall effect on elastin degradation. Vitamin D decelerates elastin degradation in vitamin D-sufficient, but not in vitamin D-deficient, COPD patients. https://bit.ly/2A9H9P7

Pseudoxanthoma elasticum (PXE) results in extensive fragmentation and calcification of elastin fibers in the peripheral arteries, which results in peripheral arterial disease (PAD). Current research focuses on the role of calcifications in the pathogenesis of PXE. Elastin degradation and calcification are shown to interact and may amplify each other. This study aims to compare plasma desmosines, a measure of elastin degradation, between PXE patients and controls and to investigate the association between desmosines and (1) arterial calcification, (2) PAD, and (3) PAD independent of arterial calcification in PXE. Plasma desmosines were quantified with liquid chromatography-tandem mass spectrometry in 93 PXE patients and 72 controls. In PXE patients, arterial calcification mass was quantified on CT scans. The ankle brachial index (ABI) after treadmill test was used to analyze PAD, defined as ABI &lt, 0.9, and the Fontaine classification was used to distinguish symptomatic and asymptomatic PAD. Regression models were built to test the association between desmosines and arterial calcification and arterial functioning in PXE. PXE patients had higher desmosines than controls (350 (290&ndash, 410) ng/L vs. 320 (280&ndash, 360) ng/L, p = 0.02). After adjustment for age, sex, body mass index, smoking, type 2 diabetes mellitus, and pulmonary abnormalities, desmosines were associated with worse ABI (&beta, (95%CI): &minus, 68 (&minus, 132, &minus, 3) ng/L), more PAD (&beta, (95%CI): 40 (7, 73) ng/L), and higher Fontaine classification (&beta, (95%CI): 30 (6, 53) ng/L), but not with arterial calcification mass. Lower ABI was associated with higher desmosines, independent from arterial calcification mass (&beta, 0.71(&minus, 1.39, 0.01)). Elastin degradation is accelerated in PXE patients compared to controls. The association between desmosines and ABI emphasizes the role of elastin degradation in PAD in PXE. Our results suggest that both elastin degradation and arterial calcification independently contribute to PAD in PXE.

Introduction: Coronavirus 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus-2. The majority of patients have at most mild symptoms, however, a significant proportion develops respiratory failure. COVID-19 may also progress beyond the lungs. Coagulopathy and thromboembolism are prevalent in severe COVID-19 and relate to decreased survival. Coagulation is an intricate balance between clot promoting and dissolving processes in which vitamin K plays a well-known role. We hypothesized that vitamin K status is reduced in patients with severe COVID-19. Methods: Vitamin K status was assessed by measuring desphospho-uncarboxylated matrix Gla protein (dp-ucMGP; inversely related to vitamin K status) and the rate of elastin degradation by measuring desmosine. We included 123 patients who were admitted with COVID-19 and 184 controls. Results: Dp-ucMGP levels were significantly elevated in COVID-19 patients (1,673Å}1,584 pmol/L) compared to controls (536±291 pmol/L; p

Breast cancer susceptibility variants frequently show heterogeneity in associations by tumor subtype1–3. To identify novel loci, we performed a genome-wide association study (GWAS) including 133,384 breast cancer cases and 113,789 controls, plus 18,908 BRCA1 mutation carriers (9,414 with breast cancer) of European ancestry, using both standard and novel methodologies that account for underlying tumor heterogeneity by estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status and tumor grade. We identified 32 novel susceptibility loci (P < 5.0 × 10−8), 15 of which showed evidence for associations with at least one tumor feature (false discovery rate (FDR)

Elastin degradation is accelerated in chronic obstructive pulmonary disease (COPD) and is partially regulated by Matrix Gla Protein (MGP), via a vitamin K-dependent pathway. The aim was to assess vitamin K status in COPD as well as associations between vitamin K status, elastin degradation, lung function parameters and mortality. A total of 192 COPD patients and 186 age-matched controls were included. In addition to this, 290 COPD patients from a second independent longitudinal cohort were also included. Vitamin K status was assessed by measuring plasma inactive MGP levels and rates of elastin degradation by measuring plasma desmosine levels. Reduced vitamin K status was found in COPD patients compared to smoking controls (p &lt, 0.0005) and controls who had never smoked (p = 0.001). Vitamin K status was inversely associated with desmosine (cohort 1: p = 0.001, cohort 2: p = 0.004). Only few significant associations between vitamin K status and lung function parameters were found. Mortality was higher in COPD patients within the quartile with the lowest vitamin K status compared to those within the other quartiles (hazard ratio 1.85, 95% confidence interval (CI), 1.21&ndash, 2.83, p = 0.005). In conclusion, we demonstrated reduced vitamin K status in COPD and an inverse association between vitamin K status and elastin degradation rate. Our results therefore suggest a potential role of vitamin K in COPD pathogenesis.

Background: The variety of LC-MS/MS methods measuring total 25(OH)D used today is vast and the comparability among these methods is still not well assessed.Methods: Here, we performed a comparison in samples of healthy donors between the currently routinely used 25(OH)D LC-MS/MS methods in the Netherlands and the Ghent University reference measurement procedure to address this issue (n = 40). Additionally, an interlaboratory comparison in patient serum samples assessed agreement between the Dutch diagnostic methods (n = 37).Results: The overall correlation of the routine methods for 25(OH)D-3 with the reference measurement procedures and with the mean of all diagnostic methods was excellent (r > 0.993 and r > 0.989, respectively). Three out of five methods aligned perfectly with both the reference measurement procedure and the median of all methods. One of the routine methods showed a small positive bias, while another showed a small negative bias consistently in both comparisons.Conclusion: The biases most probably originated from differences in calibration procedure and may be obviated by reassessing calibration of stock standards and/or calibrator matrices. In conclusion, five diagnostic centers have performed a comparison with the 25(OH)D Ghent University reference measurement procedure in healthy donor serum samples and a comparison among themselves in patient serum samples. Both analyses showed a high correlation and specificity of the routine LC-MS/MS methods, yet did reveal some small standardization issues that could not be traced back to the technical details of the different methods. Hence, this study indicates various calibration procedures can result in perfect alignment. (C) 2016 Elsevier B.V. All rights reserved.

Until recently, no prediction models for Lynch syndrome (LS) had been validated for PMS2 mutation carriers. We aimed to evaluate MMRpredict and PREMM5 in a clinical cohort and for PMS2 mutation carriers specifically. In a retrospective, clinic-based cohort we calculated predictions for LS according to MMRpredict and PREMM5. The area under the operator receiving characteristic curve (AUC) was compared between MMRpredict and PREMM5 for LS patients in general and for different LS genes specifically. Of 734 index patients, 83 (11%) were diagnosed with LS; 23 MLH1, 17 MSH2, 31 MSH6 and 12 PMS2 mutation carriers. Both prediction models performed well for MLH1 and MSH2 (AUC 0.80 and 0.83 for PREMM5 and 0.79 for MMRpredict) and fair for MSH6 mutation carriers (0.69 for PREMM5 and 0.66 for MMRpredict). MMRpredict performed fair for PMS2 mutation carriers (AUC 0.72), while PREMM5 failed to discriminate PMS2 mutation carriers from non-mutation carriers (AUC 0.51). The only statistically significant difference between PMS2 mutation carriers and non-mutation carriers was proximal location of colorectal cancer (77 vs. 28%, p

Although the spliceogenic nature of the BRCA2 c.68‐7T > A variant has been demonstrated, its association with cancer risk remains controversial. In this study, we accurately quantified by real‐time PCR and digital PCR (dPCR), the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case‐control analysis in 83,636 individuals. Co‐occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5‐fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68‐7T > A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 × 10−115. There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86–1.24) nor for a deleterious effect of the variant when co‐occurring with pathogenic variants. Our data provide for the first time robust evidence of the nonpathogenicity of the BRCA2 c.68‐7T > A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants.

Craniofrontonasal syndrome (CFNS) is an X-linked developmental malformation, caused by mutations in the EFNB1 gene, which have only been described since 2004. A genotype-phenotype correlation seems not to be present. As it is of major importance to adequately counsel patients with EFNB1 mutations and their parents, and to improve diagnosis of new patients, more information about the phenotypic features is needed. This study included 23 patients (2 male, 21 female) with confirmed EFNB1 mutations. All patients underwent a thorough physical examination and photographs were taken. If available, radiological images were also consulted. Hypertelorism, longitudinal ridging and/or splitting of nails, a (mild) webbed neck and a clinodactyly of one or more toes were the only consistent features observed in all patients. Frequently observed phenotypic features were bifid tip of the nose (91%), columellar indentation (91%) and low implantation of breasts (90%). In comparison with anthropometric data of facial proportions, patients with CFNS had a significantly different face in multiple respects. An overview of all phenotypic features is shown. Patients with EFNB1 mutations have a clear phenotype. This study will facilitate genetic counseling of parents and patients, and contribute to the diagnostic and screening process of patients with suspected CFNS. © 2014 Macmillan Publishers Limited All rights reserved.

NF1 mutations are the underlying cause of neurofibromatosis type 1 (NF1), a neuro-cardio-facio-cutaneous syndrome (NCFC). Because of the clinical overlap between NCFCs, genetic analysis of NF1 is necessary to confirm a clinical diagnosis NF1. This report describes the clinical and genetic findings of 18 years of NF1 molecular diagnostics in the Netherlands. A pathogenic mutation was found in 59.3% (1178/1985) of the index patients, mostly de novo (73.8%). The majority of the index patients (64.3%) fulfilled the National Institute of Health NF1 criteria, a pathogenic mutation was found in 80.9% of these patients. Seventy-four percent of the index patients with an NF1 pathogenic mutation and not fulfilling the NF1 criteria is

Recently a novel syndromic form of overgrowth with intellectual disability and distinct facial features was identified caused by constitutional mutations in the epigenetic regulator DNA-methyltransferase 3A (Here we present the first case of TBRS who developed AML at the age of 15 years. Whole-exome sequencing identified a constitutional heterozygousThe peculiarity of the specific R882 mutation in contrast to other

The prevalence of vitamin D deficiency in the general population has become a major public health problem. Vitamin D deficiency might have significant consequences not only to bone health but possibly to autoimmune-, infectious and cardiovascular disease. This has resulted in increased clinical testing for 25-hydroxyvitamin D (25(OH)D) in serum, as circulating 25(OH)D is regarded as the best indicator of adequate exposure to sunlight and dietary intake of vitamin D. There are reportedly over 50 vitamin D metabolites of which 25(OH)D and 1,25(OH)2D are well known to provide clinical information. More recently, there is increasing interest in measuring the C3-epimer of 25(OH)D, which has shown to contribute significantly to the 25(OH)D concentration, particularly in infant populations, and in 24,25(OH)2D, a major catabolite of 25(OH)D metabolism. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an analytical tool that allows the specific determination of all relevant vitamin D metabolites, with the potential of performing multiple analyte analysis in a single experimental setting, creating a vitamin D profile. This article reviews recent advances in the quantification of vitamin D metabolites using LC-MS/MS.

Introduction: Two RCTs suggest that vitamin D supplementation lowers exacerbation frequency in vitamin D deficient COPD patients (Lehouck, A. et al. Ann Intern Med 2012; 156:105-14/Martineau, A.R. et al. Lancet Respir Med 2015; 3:e24-5). However, information regarding the effect of vitamin D on the rate of mature elastin degradation is lacking. We hypothesized that vitamin D supplementation in vitamin D deficient (serum 25-[OH]D Methods: pDES was measured by LC-MS/MS in 142 COPD patients from the Leuven vitamin D intervention trial (100,000 IU vitamin D supplementation or placebo every 4 weeks for 1 year). Results: At baseline, no significant correlation was found between 25-[OH]D and pDES levels. No significant interaction between 25-[OH]D change and pDES change was found in the placebo arm. In the vitamin D supplementation arm, a significant negative association was found between pDES change and baseline 25-[OH]D levels (r=-0.44;p

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by a combination of neurological symptoms and hamartomatous growths, and caused by mutations in the TSC1 and TSC2 genes. Overall, TSC2 mutations are associated with a more severe disease phenotype. We identified the c. 3598C> T (R1200W) change in the TSC2 gene in seven different families. The clinical phenotypes in the families were mild, characterized by mild skin lesions, remitting epilepsy and a lack of severe mental retardation or major organ involvement. Functional analysis of the TSC2 R1200W variant, and four other TSC2 missense variants associated with a mild TSC phenotype, confirmed that the changes disrupted the TSC1-TSC2 function. Interestingly however, in each case, the TSC1-TSC2 interaction was not affected by the amino acid substitution.

Objective: Li Fraumeni syndrome (LFS) and Von Hippel-Lindau disease (VHL) are two rare hereditary tumor syndromes, characterized by a high risk of developing multiple tumors at various sites and ages for which preventive and treatment options are limited. For partners, it may be difficult to deal with the on-going threat of tumors in both their spouse and children. Therefore, this study aims to evaluate the prevalence of and factors associated with psychological distress among partners of individuals with or at high risk of LFS or VHL. Methods: As part of a nationwide, cross-sectional study, partners of individuals diagnosed with or at high risk of LFS or VHL were invited to complete a self-report questionnaire assessing distress, worries, and health-related quality of life. Results: Fifty-five (58%) of those high-risk individuals with a partner consented to having their partner approached for the study. In total, 50 partners (91%) completed the questionnaire, of whom 28% reported clinically relevant levels of syndrome-related distress. Levels of distress and worries of the partners and their high-risk spouse were significantly correlated. Younger age and a lack of social support were also associated significantly with heightened levels of distress and worries. The majority of partners (76%) believed that professional psychosocial support should be routinely offered to them. Conclusions: Approximately one-quarter of the partners exhibit clinically relevant levels of distress that warrant psychological support. The distress levels of the ‘patient’ could potentially be used to identify partners at risk of developing clinically relevant levels of distress. Copyright © 2011 John Wiley & Sons, Ltd.

Objective: The purpose of this study was to systematically analyze the associations between different TSC1 and TSC2 mutations and the neurologic and cognitive phenotype in patients with tuberous sclerosis complex (TSC). Methods: Mutation analysis was performed in 58 patients with TSC. Epilepsy variables, including EEG, were classified. A cognition index was determined based on a comprehensive neuropsychological assessment. On three-dimensional fluid-attenuated inversion recovery MR images, an automated tuber segmentation program detected and calculated the number of tubers and the proportion of total brain volume occupied by tubers (tuber/brain proportion [TBP]). Results: As a group, patients with a TSC2 mutation had earlier age at seizure onset, lower cognition index, more tubers, and a greater TBP than those with a TSC1 mutation, but the ranges overlapped considerably. Familial cases were older at seizure onset and had a higher cognition index than nonfamilial cases. Patients with a mutation deleting or directly inactivating the tuberin GTPase activating protein (GAP) domain had more tubers and a greater TBP than those with an intact GAP domain. Patients with a truncating TSC1 or TSC2 mutation differed from those with nontruncating mutations in seizure types only. Conclusions: Although patients with a TSC1 mutation are more likely to have a less severe neurologic and cognitive phenotype than those with a TSC2 mutation, the considerable overlap between both aspects of the phenotype implies that prediction of the neurologic and cognitive phenotypes in individuals with tuberous sclerosis complex should not be based on their particular TSC1 or TSC2 mutation.

In schwannomatosis, germline SMARCB1 or LZTR1 mutations predispose to the development of multiple benign schwannomas. Besides these, other tumors may occur in schwannomatosis patients. We present a 45-year-old male patient who developed multiple schwannomas and in addition a malignant type 1 papillary renal cell carcinoma (pRCC1). We identified a duplication of exon 7 of SMARCB1 on chromosome 22 in the constitutional DNA of the patient (c.796-2246_986 + 5250dup7686), resulting in the generation of a premature stop codon in the second exon 7 copy (p.Glu330*). The mutant SMARCB1 allele proved to be retained in three schwannomas and in the pRCC1 of the patient. Loss of heterozygosity analysis demonstrated partial loss of the wild-type SMARCB1 allele containing chromosome 22, suggesting loss of that chromosome in only a subset of tumor cells, in all four tumors. Immunohistochemical staining with a SMARCB1 antibody revealed a mosaic SMARCB1 expression pattern in the three benign schwannomas, but absence of expression in the malignant tumor cells of the pRCC1. To our knowledge, this difference in SMARCB1 protein expression has not been reported before. We conclude that a germline SMARCB1 mutation may predispose to the development of pRCC1, thereby further widening the spectrum of tumors that can develop in the context of schwannomatosis.

Introduction: Plasma Desmosine (pDES) reflects mature elastin degradation, and has great potential as a biomarker in COPD. One of the requirements for a COPD biomarker is short-term stability. Furthermore, there are conflicting reports regarding the influence of smoking on pDES levels in subjects without COPD. Therefore, we assessed between-day variability, and measured pDES levels in (ex-)smokers and never-smokers. Methods: pDES was measured in 16 active smokers, 26 former smokers and 34 never-smokers using liquid-chromatography-tandem mass spectrometry (LC-MS/MS) with deuterium labeled DES as internal standard. COPD was excluded with spirometry (FEV1/FVC ratio > 0.7) in (ex-)smokers. Furthermore, we measured pDES on 5 consecutive days in 15 non-smokers. Results: pDES levels were significantly higher in (ex-)smokers compared to never-smokers (p=0.01; Figure). No significant difference in pDES levels was found between active and former smokers (p=0.4). The pDES 5 day intra-person mean variation from the mean was 0.009μg/L; 7.4% (SD 0.007μg/L; 6.0%). Conclusions: pDES levels were elevated in (ex-)smokers compared to never-smokers, but no difference in pDES levels was found between active and former smokers. pDES measured with LC-MS/MS fulfilled the COPD biomarker validation requirement of short-term intra-individual stability. ![Figure][1] This study was sponsored by GlaxoSmithKline. [1]: pending:yes

Aims/hypothesis: Mitochondrial DNA (mtDNA) mutations cause several diseases, including mitochondrial inherited diabetes and deafness (MIDD), typically associated with the mtDNA A3243G point mutation on tRNALeu gene. The common hypothesis to explain the link between the genotype and the phenotype is that the mutation might impair mitochondrial metabolism expressly required for beta cell functions. However, this assumption has not yet been tested. Methods: We used clonal osteosarcoma cytosolic hybrid cells (namely cybrids) harbouring mitochondria derived from MIDD patients and containing either exclusively wild-type or mutated (A3243G) mtDNA. According to the importance of mitochondrial metabolism in beta cells, we studied the impact of the mutation on key parameters by comparing stimulation of these cybrids by the main insulin secretagogue glucose and the mitochondrial substrate pyruvate. Results: Compared with control mtDNA from the same patient, the A3243G mutation markedly modified metabolic pathways leading to a high glycolytic rate (2.8-fold increase), increased lactate production (2.5-fold), and reduced glucose oxidation (−83%). We also observed impaired NADH responses (−56%), negligible mitochondrial membrane potential, and reduced, only transient ATP generation. Moreover, cybrid cells carrying patient-derived mutant mtDNA exhibited deranged cell calcium handling with increased cytosolic loads (1.4-fold higher), and elevated reactive oxygen species (2.6-fold increase) under glucose deprivation. Conclusions/interpretation: The present study demonstrates that the mtDNA A3243G mutation impairs crucial metabolic events required for proper cell functions, such as coupling of glucose recognition to insulin secretion

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation and the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. Here we describe a deletion encompassing the TSC1 gene and two neighboring transcripts on chromosome 9q34 in six affected individuals from a family with TSC. To our knowledge, this is the first report of such a large deletion at the TSC1 locus and indicates that screening for similar mutations at the TSC1 locus is warranted in individuals with TSC.

Contains fulltext : 57795.pdf (Publisher’s version ) (Closed access) The overall rate of an ipsilateral breast tumour recurrence (IBTR) after breast-conserving therapy (BCT) ranges from 1% to 2% per year. Risk factors include young age but data on the impact of BRCA1/2 mutations or a definite positive family history for breast cancer are scarce. We investigated IBTR after BCT in patients with hereditary breast cancer (HBC). Through our family cancer clinic we identified 87 HBC patients, including 26 BRCA1/2 carriers, who underwent BCT between 1980 and 1995 (cases). They were compared to 174 patients with sporadic breast cancer (controls) also treated with BCT, matched for age and year of diagnosis. Median follow up was 6.1 years for the cases and 6.0 years for controls. Patient and tumour characteristics were similar in both groups. An IBTR was observed in 19 (21.8%) hereditary and 21 (12.1%) sporadic patients. In the hereditary patients more recurrences occurred elsewhere in the breast (21% versus 9.5%), suggestive of new primaries. Overall, the actuarial IBTR rate was similar at 2 years, but higher in hereditary as compared to sporadic patients at 5 years (14% versus 7%) and at 10 years (30% versus 16%) (P=0.05). Post-relapse and overall survival was not different between hereditary and sporadic cases. Hereditary breast cancer was therefore associated with a higher frequency of early (2-5 years) and late (>5 years) local recurrences following BCT. These data suggest an indication for long-term follow up in HBC and should be taken into account when additional 'risk-reducing' surgery after primary BCT is eventually considered.

This review provides a compact overview on the contribution of mutations in mtDNA to the pathogenesis of diabetes mellitus, with emphasis on the A3243G mutation in the tRNA(Leu, UUR) gene. This mutation associates in most individuals with maternally inherited diabetes and deafness (MIDD) whereas in some other carriers the MELAS syndrome or a progressive kidney failure is seen. Possible pathogenic mechanisms are discussed especially the question why particular mutations in mtDNA associate with distinct clinical entities. Mutations in mtDNA can affect the ATP production, thereby leading to particular clinical phenotypes such as muscle weakness. On the other hand mtDNA mutations may also alter the intracellular concentration of mitochondrial metabolites which can act as signalling molecules, such as Ca or glutamate. This situation may contribute to the development of particular phenotypes that are associated with distinct mtDNA mutations.

In 517 Dutch families at a family cancer clinic, we screened for BRCA1/2 alterations using the Protein Truncation Test (PTT) covering approximately 60% of the coding sequences of both genes and direct testing for a number of previously identified Dutch recurrent mutations. In 119 (23%) of the 517 families, we detected a mutation in BRCA1 (n=98; 19%) or BRCA2 (n=21; 4%). BRCA1/2 mutations were found in 72 (52%) of 138 families with breast and ovarian cancer (HBOC), in 43 (13%) of the 339 families with breast cancer only (HBC), in 4 (36%) of 11 families with ovarian cancer only (HOC), and in nine of 29 families with one single young case (

PURPOSE: Women with a high breast cancer risk due to a familial predisposition may choose between preventive surgery and regular surveillance. The effectiveness of surveillance in high-risk women and especially BRCA1/2 mutation carriers is unknown. We present first results from a single large family cancer clinic. PATIENTS AND METHODS: Women with breast cancer risk over 15% were examined by physical examination every 6 months and mammography every year. Detection rates and screening parameters were calculated for the total group and separately for different age and genetic risk groups. RESULTS: At least one examination was performed in 1,198 women: 449 moderate and 621 high-risk women and 128 BRCA1/2 mutation carriers. Within a median follow-up of 3 years, 35 breast cancers were detected (four ductal carcinoma-in-situ; 31 invasive tumors); the average detection rate was 9.7 per 1,000. Detection rates (95% confidence interval) for moderate and high-risk women and BRCA1/2 carriers were 3.3 (1.1 to 8.6), 8.4 (5.4 to 13.2), and 33 (17 to 63) per 1,000 person-years, respectively. The ratio of observed cases versus breast cancers expected in an average-risk population of comparable age was 2.7, 7.0 and 23.7 respectively. Overall, node negativity was 65%; 34% of primary tumors were less than 10 mm; sensitivity was 74%. Results with respect to tumor stage and sensitivity were less favorable in BRCA1/2 carriers and in women under the age of 40. CONCLUSION: It is possible to identify young women at high risk for breast cancer. The number of cancers detected was significantly greater than expected in an age-matched average-risk population and related to the risk category. Overall, screening parameters were comparable to population screening data, with less favorable results in the youngest age group (< 40) and BRCA1/2 carriers.

SUMMARY Aim Maternally inherited diabetes and deafness (MIDD) associates with a mutation at position 3243 in mitochondrial DNA. Phenotypic expression of MIDD includes Type 1-like and Type 2-like diabetes. This study examined whether HLA-DQ phenotype and the degree of heteroplasmy in leucocyte and oral mucosa DNA influence clinical expression of the 3242 mutation. Methods In a group of 20 unrelated probands with MIDD, eight with Type 1-like diabetes, 12 with Type 2-like diabetes, HLA-DQ type and degree of heteroplasmy for the 3243 mutation were determined. HLA-DQA1/DQB1 phenotypes were categorized as predisposing, neutral or protective for autoimmune-mediated Type 1 diabetes. Results No differences were observed between Type 1 and Type 2-like MIDD groups with respect to the cumulative frequency of protective and predisposing HLA-DQ types. Predisposing HLA-DQ types are more prevalent in MIDD patients than in the control population (P

PURPOSE: Breast cancer in BRCA1 and BRCA2 gene-mutation carriers may differ from so-called sporadic breast cancer in clinical features and behavior. These potential differences may be of importance for the prevention, screening, and, ultimately, treatment of breast cancer in women with such germline mutations. Thus far, there have been very few studies on the survival of BRCA2-associated breast cancer patients. PATIENTS AND METHODS: We determined the disease-free and overall survival of 28 breast cancer patients from 14 consecutive families with eight different BRCA2 germline mutations. These patients' survival and tumor characteristics were compared with those of a control group of 112 sporadic breast cancer patients matched to them by age and year of diagnosis. RESULTS: The 5-year disease-free survival was 52% for each group (P = .91); the overall survival was 74% for BRCA2 carriers and 75% for sporadic cases (P = .50). At the time of diagnosis, tumors from the BRCA2 carriers were borderline significantly larger in comparison to the tumors in sporadic cases (P = .05), but axillary nodal status was not significantly different in the two groups (node-negativity, 63% v 52.8%, respectively; P = .34). With respect to steroid receptor status, BRCA2-associated tumors were more likely to be steroid receptor-positive, especially regarding progesterone receptor status (100% v 76.7% positive, respectively; P = .06). Stage-adjusted recurrence and death rates were nonsignificantly better for BRCA2 cases (hazard ratios of 0.84 and 0.59 [P = .61 and P = .19], respectively). In contrast, after 5 years, the rate of metachronous contralateral breast cancer in BRCA2 patients was 12% (v 2% in controls; P = .02). CONCLUSION: Patients with hereditary breast cancer due to BRCA2 have a similar prognosis when compared with age-matched sporadic breast cancer patients. Contrary to our previous observation regarding BRCA1-associated breast cancer, BRCA2 tumors tended to be steroid receptor-positive, instead of steroid receptor-negative.

A 12-year-old boy with tuberous sclerosis complex (TSC) presented with a large retroperitoneal tumour. Exploratory surgery revealed an infiltrative tumour originating from the pancreas, with local metastases to the lymph nodes. The histologal diagnosis was a malignant islet cell tumour. Retrospectively measured pancreatic hormone levels, however, were normal. A connection between the malignancy and TSC was demonstrated by loss of heterozygosity of the TSC2 gene in the tumour. The primary mutation Q478X in this patient was identified in exon 13 of the TSC2 gene on chromosome 16. Conclusion Pancreatic islet cell tumours have been mainly associated with multiple endocrine neoplasia syndrome type 1. In our case we demonstrate a direct relationship of this tumour to tuberous sclerosis complex, in the absence of further signs of multiple endocrine neoplasia syndrome type 1.

Congenital bilateral absence of the vas deferens (CBAVD) is found in 1-2% of infertile males and in most male cystic fibrosis (CF) patients. CF and some of the CBAVD cases were found to share the same genetic background. In this study, 21 males with CBAVD had extensive physical and laboratory testing for symptoms of CF. Possible defective cellular chloride transport was measured by interstitial current measurement of rectal suction biopsies. Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis was performed for 10 common CFTR mutations. CF-related symptoms were found in six men. On laboratory testing slightly abnormal liver and pancreatic function was found in seven patients. The sweat test was found to be abnormal in four patients; interstitial current measurement showed defective chloride excretion in 11 patients. CFTR gene mutations were found in 66% of the patients: eight were compound heterozygotes; in six, only one common mutation could be detected. The 5T allele in one copy of intron 8 was found in four men. CBAVD appears to be a heterogeneous clinical and genetic condition. A CFTR gene mutation was found in both copies of the allele or interstitial current measurement showed defective chloride excretion in 14/21 cases. Genetic counselling is clearly indicated for couples seeking pregnancy through epididymal or testicular sperm aspiration and intracytoplasmic sperm injection.

Hereditary breast cancer has been associated with mutations in the BRCA1 and BRCA2 genes and has a natural history different from sporadic breast cancer. We investigated disease-free and overall survival for patients with a proven BRCA1 alteration. We estimated disease-free and overall survival for 49 Dutch patients from 19 consecutive families with a proven specific BRCA1 mutation and one family with strong evidence for linkage to the BRCA1 gene. We compared clinical outcome and data on tumour size, histology, axillary nodal status, contralateral breast cancer, and oestrogen-receptor and progesterone-receptor status with those of 196 patients with sporadic breast cancer, matched for age and year of diagnosis. Disease-free survival for BRCA1 and sporadic patients at 5 years was 49% (95% CI 33-64) and 51% (43-59), respectively (p=0.98). Overall survival at 5 years was 63% (47-76) and 69% (62-76), respectively (p=0.88). Recurrence and death rates did not differ significantly between groups. Hazard ratios for recurrence and death among BRCA1 patients were 1.00 (0.65-1.55) and 1.04 (0.63-1.71) relative to sporadic patients (p=0.88), and these did not differ significantly after adjustment for prognostic factors. Patients with BRCA1-associated breast cancer had twice as many progesterone-receptor-negative tumours (p