Here we present an ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method for extracellular measurements of known and unexpected metabolites in parallel. The method was developed ...by testing 86 metabolites, including amino acids, organic acids, sugars, purines, pyrimidines, vitamins, and nucleosides, that can be resolved by combining chromatographic and
m
/
z
dimensions. Subsequently, a targeted quantitative method was developed for 80 metabolites. The presented method combines a UPLC approach using hydrophilic interaction liquid chromatography (HILIC) and MS detection achieved by a hybrid quadrupole–time of flight (Q–ToF) mass spectrometer. The optimal setup was achieved by evaluating reproducibility and repeatability of the analytical platforms using pooled quality control samples to minimize the drift in instrumental performance over time. Then, the method was validated by analyzing extracellular metabolites from acute lymphoblastic leukemia cell lines (MOLT-4 and CCRF-CEM) treated with direct (A-769662) and indirect (AICAR) AMP activated kinase (AMPK) activators, monitoring uptake and secretion of the targeted compound over time. This analysis pointed towards a perturbed purine and pyrimidine catabolism upon AICAR treatment. Our data suggest that the method presented can be used for qualitative and quantitative analysis of extracellular metabolites and it is suitable for routine applications such as in vitro drug screening.
Figure
UPLC-MS analysis of extracellular metabolites from acute lymphoblastic leukemia cell lines treated with AMP activated kinase (AMPK) activators points out that purine catabolism is affected upon AICAR treatment.
Adult human height is one of the classical complex human traits. We searched for sequence variants that affect height by scanning the genomes of 25,174 Icelanders, 2,876 Dutch, 1,770 European ...Americans and 1,148 African Americans. We then combined these results with previously published results from the Diabetes Genetics Initiative on 3,024 Scandinavians and tested a selected subset of SNPs in 5,517 Danes. We identified 27 regions of the genome with one or more sequence variants showing significant association with height. The estimated effects per allele of these variants ranged between 0.3 and 0.6 cm and, taken together, they explain around 3.7% of the population variation in height. The genes neighboring the identified loci cluster in biological processes related to skeletal development and mitosis. Association to three previously reported loci are replicated in our analyses, and the strongest association was with SNPs in the ZBTB38 gene.
The common sequence variants that have recently been associated with cancer risk are particular to a single cancer type or at most two. Following up on our genome-wide scan of basal cell carcinoma, ...we found that rs401681C on chromosome 5p15.33 satisfied our threshold for genome-wide significance (OR = 1.25, P = 3.7 × 10−12). We tested rs401681 for association with 16 additional cancer types in over 30,000 cancer cases and 45,000 controls and found association with lung cancer (OR = 1.15, P = 7.2 × 10−8) and urinary bladder, prostate and cervix cancer (ORs = 1.07−1.31, all P < 4 × 10−4). However, rs401681C seems to confer protection against cutaneous melanoma (OR = 0.88, P = 8.0 × 10−4). Notably, most of these cancer types have a strong environmental component to their risk. Investigation of the region led us to rs2736098A, which showed stronger association with some cancer types. However, neither variant could fully account for the association of the other. rs2736098 corresponds to A305A in the telomerase reverse transcriptase (TERT) protein and rs401681 is in an intron of the CLPTM1L gene.
Background Mutations in the BRCA2 gene are associated with an increased risk of prostate cancer, but it is not known whether they are associated with progression of the disease. We compared prostate ...cancer–specific survival, disease stage, and tumor grade between prostate cancer patients carrying the Icelandic BRCA2 999del5 founder mutation and noncarriers. Methods Using population-based registries, we identified all 596 prostate cancer patients who were diagnosed in Iceland during 1955 through 2004 among 29603 male relatives of unselected breast cancer probands. BRCA2 mutation status could be determined for 527 patients (88.4%). Stage and grade were abstracted from original records, blindly with respect to mutation status, for a subgroup of 89 patients that included all mutation carriers and, for each carrier, two control patients without the BRCA2 999del5 mutation who were matched to the carrier on years of diagnosis and birth. Hazard ratios (HRs) and 95% confidence intervals (CIs) for prostate cancer–specific survival were estimated using multivariable regression models. All statistical tests were two-sided. Results The mutation was carried by 30 patients (5.7%). Compared with noncarriers, BRCA2 999del5 mutation carriers had a lower mean age at diagnosis (69.0 years versus 74.0 years; P = .002), more advanced tumor stage (stages 3 or 4, 79.3% versus 38.6%; P<.001), higher tumor grade (grades G3–4, 84.0% versus 52.7%, P = .007), and shorter median survival time (2.1 years, 95% CI = 1.4 to 3.6 years, versus 12.4 years, 95% CI = 9.9 to 19.7 years). Carrying the BRCA2 999del5 mutation was also associated with an increased risk of dying from prostate cancer (adjusting for year of diagnosis and birth, HR = 3.42, 95% CI = 2.12 to 5.51); the association remained after adjustment for stage and grade (HR = 2.35, 95% CI = 1.08 to 5.11). The prognosis of BRCA2 999del5 mutation carriers was not associated with period of diagnosis or with relatedness to breast cancer probands. Conclusions The Icelandic BRCA2 999del5 founder mutation was strongly associated with rapidly progressing lethal prostate cancer.
Background and Objectives
Non‐invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental ...consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non‐invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi‐national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations.
Materials and Methods
We reviewed the literature covering validation for in‐vitro diagnostic (IVD) assays in general and for non‐invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co‐authors.
Results
In relation to Annex VIII of the In‐Vitro‐Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non‐invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti‐D prophylaxis for non‐immunized RhD negative women should be placed under risk class C. To ensure high quality of non‐invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre‐analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed.
Conclusion
These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in‐house assays.
To search for new sequence variants that confer risk of cutaneous basal cell carcinoma (BCC), we conducted a genome-wide SNP association study of 930 Icelanders with BCC and 33,117 controls. After ...analyzing 304,083 SNPs, we observed signals from loci at 1p36 and 1q42, and replicated these associations in additional sample sets from Iceland and Eastern Europe. Overall, the most significant signals were from rs7538876 on 1p36 (OR = 1.28, P = 4.4 × 10−12) and rs801114 on 1q42 (OR = 1.28, P = 5.9 × 10−12). The 1p36 locus contains the candidate genes PADI4, PADI6, RCC2 and ARHGEF10L, and the gene nearest to the 1q42 locus is the ras-homolog RHOU. Neither locus was associated with fair pigmentation traits that are known risk factors for BCC, and no risk was observed for melanoma. Approximately 1.6% of individuals of European ancestry are homozygous for both variants, and their estimated risk of BCC is 2.68 times that of noncarriers.
► This is the first metabolomic study reporting analytical protocols for platelets. ► Seven different extraction methods and two different UPLC–MS methods were tested. ► MeOH–H2O extraction coupled ...with HILIC–MS method identified 107 metabolites.
An extraction method for intracellular metabolite profiling should ideally be able to recover the broadest possible range of metabolites present in a sample. However, the development of such methods is hampered by the diversity of the physico-chemical properties of metabolites as well as by the specific characteristics of samples and cells. In this study, we report the optimization of an UPLC–MS method for the metabolite analysis of platelet samples. The optimal analytical protocol was determined by testing seven different extraction methods as well as by employing two different LC–MS methods, in which the metabolites were separated by using hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC). The optimal conditions were selected using the coverage of the platelets’ metabolome, the response of the identified metabolites, the reproducibility of the analytical method, and the time of the analysis as main evaluation criteria. Our results show that methanol–water (7:3) extraction coupled with HILIC–MS method provides the best compromise, allowing identification of 107 metabolites in a platelet cell extract sample, 91% of them with a RSD% lower than 20. A higher number of metabolites could be detected when analyzing the platelet samples with two different LC–MS methods or when using complementary extraction methods in parallel.
Background and Objectives
Fetal RHD genotyping of cell‐free fetal DNA from RhD‐negative pregnant women can be used to guide targeted antenatal and postnatal anti‐D prophylaxis for the prevention of ...RhD immunization. To assure the quality of clinical testing, we conducted an external quality assessment workshop with the participation of 28 laboratories.
Materials and Methods
Aliquots of pooled maternal plasma were sent to each laboratory. One sample was positive, and the second sample was negative for fetal RHD, verified by pre‐workshop testing using quantitative real‐time PCR (qPCR) analysis of RHD exons 4, 5, 7 and 10. Plasma samples were shipped at room temperature. A reporting scheme was supplied for data collection, including questions regarding the methodological setup, results and clinical recommendations. Different methodological approaches were used, all employing qPCR with a total of eight different combinations of RHD exon targets. The samples were tested blindly.
Results
Fetal RHD genotyping was performed with no false‐negative and no false‐positive results. One inconclusive result was reported for the RHD‐positive sample, and four inconclusive results were reported for the RHD‐negative sample. All clinical conclusions were satisfactory.
Conclusion
This external quality assessment workshop demonstrates that despite the different approaches taken to perform the clinical assays, fetal RHD genotyping is a reliable laboratory assay to guide targeted use of Rh prophylaxis in a clinical setting.
Estimates of an 80–90% risk of breast cancer for carriers of germline mutations in the
BRCA1 and
BRCA2 genes are based on studies of families at high risk of breast cancer. Risk estimates for a ...population are possible if the mutation status of a representative sample of that population can be assessed. In Iceland, one common founder
BRCA2 mutation occurs in 0·6% of the population. Iceland has a population-based cancer registry and a large collection of pedigrees, and estimation of cancer risk in mutation carriers is therefore possible.
We studied 575 breast-cancer patients, 541 women and 34 men unselected for family history of breast cancer. Data on cancer in first-degree relatives were available from the cancer registry. Risk of cancer was estimated by comparing the history of cancer in first-degree relatives of carriers and non-carriers.
56 (10·4%) of the 541 women and 13 (38%) of the 34 men carried the 999del5 mutation. The estimated risk of breast cancer at age 50 for all female carriers of the 999del5 mutation was 17·0% (95% CI 9·1–25·9) and 37·2% (22·4–53·9) at age 70.
The results of our population-based study show that the mean risk of breast cancer in carriers of mutation in
BRCA2 is lower than previously suggested. Individual risk assessment will, however, have to take account of family history.
The BRCA2 gene on chromosome 13 has been shown to be associated with familial male and female breast cancer. Here we describe a study on BRCA2 in 21 Icelandic families, including 9 with male breast ...cancer. We have previously reported linkage to the BRCA2 region in an Icelandic male breast cancer family and subsequently found a strong indication of linkage to BRCA2 and the same BRCA2 haplotype in breast cancer cases from 15 additional families, indicating a common origin. We describe a five base-pair deletion in exon 9 of BRCA2 in an affected male from the male breast cancer family. The same mutation occurs in all the families with the shared BRCA2 haplotype indicating a founder effect. Among mutation carriers there are 12 males with breast cancer, which accounts for 40% of all males diagnosed with breast cancer in Iceland over the past 40 years. Three of them have no family history of breast cancer indicating that this mutation may have variable penetrance. The same BRCA2 mutation appears to be associated with different cancer phenotypes in this population including male and female breast cancer, prostate cancer, pancreas cancer and ovarian cancer.
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