Lessons from 10 years' experience running the Fiom KID-DNA database, a voluntary DNA-linking register for donor-conceived people and donors in The Netherlands.

Worldwide, there is increasing acknowledgment of the importance of getting access to ancestry information. More and more countries facilitate access to this information through law changes and voluntary contact-services. In the Netherlands, the state-funded Fiom KID-DNA database was established in 2010 to facilitate information and/or contact exchange between those people who are genetically related as a result of donor-assisted conception. By the end of 2021, 846 donors and 2355 donor-conceived people are registered in the database. For 25% of the donors a link was found with one or more donor-conceived people, and 39% of the donor-conceived people were linked to a donor-profile. Fiom offers support by professionally qualified staff throughout the entire process from registration to contact to donor-conceived people, donors and their relatives. During the period of more than 10 years several challenges emerged; how does a state-funded DNA database function in the area of commercial DNA databases?; what can be learned from the continuous growing donor-conceived half-siblings networks?; how to deal with malpractices from the past and how to cope with ageing donors?

DNA hypermethylation analysis in sputum of asymptomatic subjects at risk for lung cancer participating in the NELSON trial: argument for maximum screening interval of 2†years.

AIMS: Lung cancer is the major contributor to cancer mortality due to metastasised disease at time of presentation. The current study investigated DNA hypermethylation of biomarkers RASSF1A, APC, cytoglobin, 3OST2, FAM19A4, PHACTR3 and PRDM14 in sputum of asymptomatic high-risk individuals from the NELSON lung cancer low-dose spiral CT screening trial to detect lung cancer at preclinical stage. METHODS: Subjects were selected with (i) lung cancer in follow-up (cases; n=65), (ii) minor cytological aberrations (controls; n=120) and (iii) a random selection of subjects without cytological aberrations (controls; n=99). Median follow-up time for controls was 80 months. Cut-off values were based on high specificity to assess diagnostic value of the biomarkers. RESULTS: RASSF1A may denote presence of invasive cancer because of its high specificity (93% (95% CI 89% to 96%); sensitivity 17% (95% CI 4% to 31%), with best performance in a screening interval of 2 years. The panel of RASSF1A, 3OST2 and PRDM14 detected 28% (95% CI 11% to 44%) of lung cancer cases within 2 years, with specificity of 90% (95% CI 86% to 94%). Sputum cytology did not detect any lung cancers. CONCLUSIONS: In a lung cancer screening setting with maximum screening interval of 2 years, DNA hypermethylation analysis in sputum may play a role in the detection of preclinical disease, but complementary diagnostic markers are needed to improve sensitivity.

SF3B1 and EIF1AX mutations occur in primary leptomeningeal melanocytic neoplasms; yet another similarity to uveal melanomas.

INTRODUCTION: Like uveal melanomas, primary leptomeningeal melanocytic neoplasms (LMNs) frequently carry GNAQ and GNA11 mutations. However, it is currently unknown whether these LMNs harbor mutations in BAP1, SF3B1 and/or EIF1AX like uveal melanomas as well. In this study, we used Sanger sequencing for the detection of mutations in SF3B1 (hotspots in exon 14 and 15) and EIF1AX (exon 1 and 2 and flanking intronic regions) in a series of 24 primary LMNs. Additionally, BAP1 immunohistochemistry was used as a surrogate marker for the detection of inactivating mutations in the BAP1 gene. RESULTS: Mutations in either SF3B1 or EIF1AX were identified in 8 out of 24 primary LMNs (33 %). The presence of these mutations was mutually exclusive and occurred in primary LMNs of different malignancy grades (melanocytomas, intermediate-grade melanocytic tumors, melanomas). Complete absence of nuclear BAP1 staining as is typically seen in BAP1-mutated tumors was not observed. CONCLUSIONS: Our finding that an SF3B1 or EIF1AX mutation is present in a substantial subset of primary LMNs underscores that these tumors genetically resemble uveal melanoma and are different from cutaneous melanoma at the genetic level. This information may not only aid in the differential diagnosis of primary versus metastatic melanocytic tumor in/around the central nervous system, but also in the identification of more promising therapeutic approaches targeting the molecular pathways involved in the oncogenesis of LMNs. As none of the primary LMNs in our series showed complete loss of nuclear BAP1 protein, it is unlikely that BAP1 mutations are frequent in these tumors but the role of this gene warrants further investigation.

Mutated alleles of the rod and cone Na-Ca+K-exchanger genes in patients with retinal diseases.

PURPOSE: To study the possible involvement of the rod (SLC24A1) and cone (SLC24A2) Na-Ca+K exchanger (NCKX) genes in retinal diseases. METHODS: DNA was collected from unrelated patients with retinal disease, mainly from North America. A human genomic library was screened with the cone NCKX cDNA, and hybridizing clones were sequenced to determine the genomic organization of the SLC24A2 gene. The single-strand conformation polymorphism (SSCP) technique and direct sequencing were used to screen the patients' DNA for mutations in SLC24A1 and SLC24A2. The effect of selected missense changes on protein function was tested by measuring potassium-dependent Na-Ca exchange of the mutant proteins expressed in insect cells. RESULTS: Twenty-seven novel sequence changes were found in the rod NCKX gene, 21 of which are unlikely to be pathogenic, because they did not cosegregate with the disease or did not affect conserved regions of the protein. Of the remaining six, two were frameshift mutations found in one patient each. If translated, these alleles would encode nonfunctional proteins. Three of the six possibly pathogenic mutations were missense changes located in conserved regions, and their protein functions were assayed. Only one (Ile992Thr) had a significantly low level of exchanger function, but it was found in two unrelated patients who were heterozygotes with different retinal diseases, and this mutation could not be unequivocally associated with either disease. The last of the six changes is likely to create a new splice acceptor site. The genomic organization of the cone NCKX gene was determined, and it contained 11 exons with a few splice variants. Fifteen novel sequence changes were identified in the cone exchanger gene in patients with a cone dysfunction or degeneration. Only three of these sequence changes, all missense changes found in heterozygous patients, were considered possibly pathogenic. Functional analysis showed only a slight reduction in the activity of the corresponding mutant proteins. CONCLUSIONS: Although variant alleles of the rod and cone NCKX genes were found, none could be definitively associated with a specific retinal disease. The human phenotype associated with mutant exchanger alleles remains unknown.

Microarray as a model for quantitative visualization chemistry.

For visualization of proteins or nucleic acids, direct and indirect in situ fluorescence and absorption methods (immunohistochemistry and cytochemistry) have existed for many years. The authors describe a new experimental approach using microarray as a model to quantitatively compare both visualization methods. The spots obtained with the microarray robot had a progressive twofold decrease in concentrations and are used as objects with known amounts of DNA. Subsequent hybridization resulted in a direct fluorescence (DF) label or in hapten for indirect fluorescence (IF) and absorption. The results show that the image of the object in the IF method is larger than that in the DF method because of an edge effect, with stronger staining at the circumference. This leads to a higher plateau level and an 8- to 10-fold reduction in the detection threshold for IF compared with DF. These features are especially useful for one-color DNA-related microarray analysis, such as single nucleotide polymorphism, loss of heterozygosity, and mutation analysis, provided that the spots are not designed directly adjacent to each other, so that the edge effect is taken into account. The slope of the linear range for the IF method is much steeper than for the DF method, pointing to a narrow dynamic range in immunohistochemistry. It is noteworthy that the detection limit for absorption images after indirect immunoenzyme visualization is lower than for the DF images. The indirect immunohistochemistry semiquantitative absorption signal was at least similar compared with the DF fluorescence. In conclusion, an explanation for the difficulties experienced in quantitative immunohistochemistry is provided, and the data emphasize that in general, for daily pathology, semiquantitative patterns should suffice. Indirect labeling of DNA has useful characteristics for application in microarray analyses because of the large signal enhancement.

The retinal rod and cone Na+/Ca2+-K+ exchangers.

The past few years has seen significant progress in our understanding of the retinal rod and cone Na+/Ca2+-K+ exchanger (NCKX) genes. The human rod and cone NCKX genes were localized to chromosomes 15q22 and 9p22, respectively. In situ hybridization localized the rod and cone NCKX transcripts in both human and chicken retinas: rod NCKX transcripts were found only in the inner segments of rods, whereas cone NCKX transcripts were found in a subset of retinal ganglion cells as well as in the inner segments of cones. We identified two sets of putative transmembrane spanning segments (TM's) as the only sequence elements strongly conserved between the rod and cone NCKX cDNAs, as well as between mammalian NCKX cDNAs and NCKX cDNAs cloned from lower organisms (C. elegans and Drosophila). The two sets of TM's make up less than onethird of the rod NCKX sequence and less than half of the cone NCKX sequence. Basic cation binding properties as inferred from an analysis of 45Ca transport rates and NCKX currents were very similar for all our NCKX clones, implying that conserved residues within the two sets of TM's contain all the residues involved in cation binding and cation transport.

Methylation analysis in spontaneous sputum for lung cancer diagnosis.

OBJECTIVES: Lung cancer is the most fatal cancer in the developed world due to presence of metastases at time of diagnosis. The aim of this study is to examine DNA hypermethylation in sputum compared to sputum cytology for the diagnosis of lung cancer. A novel risk analysis is introduced, using the distinction between diagnostic and risk markers. METHODS: Two independent sets were randomly composed from a prospectively collected sputum bank (Set 1: n = 98 lung cancer patients, n = 90 controls; Set 2: n = 60 lung cancer patients, n = 445 controls). Sputum cytology was performed for all samples. The following DNA hypermethylation markers were tested in both sets: RASSF1A, APC and cytoglobin (CYGB). Two statistical analyses were conducted: multivariate logistic regression and a risk classification model based on post-test probabilities. RESULTS: In multivariate analysis, RASSF1A was the best of the three markers in discriminating lung cancer cases from controls in both sets (sensitivity 41-52%, specificity 94-96%). The risk model showed that 36% of lung cancer patients were defined as "high risk" (≥ 60% chance on lung cancer) based on RASSF1A hypermethylation in Set 1. The model was reproducible in Set 2. Risk markers (APC, CYGB) have less diagnostic value. Sensitivity of cytology for lung cancer diagnosis was 22%. RASSF1A hypermethylation yielded a sensitivity of 45%. The combined sensitivity for RASSF1A with cytological diagnosis increased to 52% with similar specificity (94%). CONCLUSION: In a diagnostic setting, hypermethylation analysis in sputum is possible when a diagnostic marker is used. However, risk markers are insufficient for this purpose.

Diagnosing peripheral lung cancer: the additional value of the Ras-association domain family 1A gene methylation and Kirsten rat sarcoma 2 viral oncogene homolog mutation analyses in washings in nondiagnostic bronchoscopy.

BACKGROUND: The diagnostic yield of bronchoscopy in patients with endoscopically nonvisible (peripheral) tumors varies from 40% to 56%. Increasingly, molecular markers in bronchial washings are being investigated to improve the diagnostic yield. The aim of this study was to analyze the diagnostic value of the Ras association domain family 1A gene (RASSF1A) methylation analysis in washings in nondiagnostic bronchoscopy in the analysis of patients with suspected lung cancer who had peripheral tumors. Furthermore, the additional diagnostic value of Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) mutations with RASSF1 methylation was analyzed. METHODS: From a prospectively collected series, 129 patients with lung cancer and 28 control subjects were analyzed retrospectively regarding the methylation status of the promoter region of the RASSF1A gene by quantitative methylation-specific polymerase chain reaction and KRAS point mutations by using the sensitive Point-EXACCT method. RESULTS: A total of 40% of the lung cancer patients had peripheral tumors, and 17 patients had a nondiagnostic bronchoscopy. In these patients, RASSF1A methylation was detected in the washings of four patients (24%), and KRAS mutations were detected in the washings of two patients (12%). In total, 29% of the false-negative or doubtful cytology results were accompanied by RASSF1A methylation or KRAS mutation results that were highly suggestive of malignancy. The proportion of RASSF1A methylation was significantly higher in central and larger tumors. No relevant RASSF1A methylation was detected in control samples. CONCLUSIONS: Our data suggest that the molecular analysis of two biomarkers in nondiagnostic bronchial washings may better guide diagnostic procedures in patients with suspected lung cancer.

EGFR and KRAS quality assurance schemes in pathology: generating normative data for molecular predictive marker analysis in targeted therapy.

INTRODUCTION: The aim of this study was to compare the reproducibility of epidermal growth factor receptor (EGFR) immunohistochemistry (IHC), EGFR gene amplification analysis, and EGFR and KRAS mutation analysis among different laboratories performing routine diagnostic analyses in pathology in The Netherlands, and to generate normative data. METHODS: In 2008, IHC, in-situ hybridisation (ISH) for EGFR, and mutation analysis for EGFR and KRAS were tested. Tissue microarray sections were distributed for IHC and ISH, and tissue sections and isolated DNA with known mutations were distributed for mutation analysis. In 2009, ISH and mutation analysis were evaluated. False-negative and false-positive results were defined as different from the consensus, and sensitivity and specificity were estimated. RESULTS: In 2008, eight laboratories participated in the IHC ring study. In only 4/17 cases (23%) a consensus score of ≥75% was reached, indicating that this analysis was not sufficiently reliable to be applied in clinical practice. For EGFR ISH, and EGFR and KRAS mutation analysis, an interpretable result (success rate) was obtained in ≥97% of the cases, with mean sensitivity ≥96% and specificity ≥95%. For small sample proficiency testing, a norm was established defining outlier laboratories with unsatisfactory performance. CONCLUSIONS: The result of EGFR IHC is not a suitable criterion for reliably selecting patients for anti-EGFR treatment. In contrast, molecular diagnostic methods for EGFR and KRAS mutation detection and EGFR ISH may be reliably performed with high accuracy, allowing treatment decisions for lung cancer.

Circulating DNA is a non-invasive prognostic factor for survival in non-small cell lung cancer.

INTRODUCTION: Circulating plasma DNA is present in a considerably higher concentration in lung cancer patients than in controls. Conflicting data are reported about circulating DNA as a prognostic factor. The aim of this study was to prospectively analyse the relationship of circulating plasma DNA with overall survival (OS) of previously untreated non-small cell lung cancer (NSCLC) patients. METHODS: 46 untreated NSCLC patients and 21 controls with a follow-up time of 6.5 years were analyzed. Quantification of baseline circulating plasma DNA was performed by a real-time quantitative polymerase chain reaction (qPCR) targeting the human beta-globin gene. Survival analysis was performed using the Kaplan-Meier method and compared with a Cox-regression analysis. RESULTS: The median DNA concentration of the patients who died (87%) was significantly higher compared to the patients that survived at the end of follow-up (55ng/ml versus 23ng/ml, p=0.02). In patients with higher DNA concentration overall survival was significantly worse. In this study no relation of DNA concentration with tumour characteristics, age, gender or pulmonary inflammatory conditions was found. CONCLUSION: In this study a high circulating plasma DNA concentration at time of diagnosis in NSCLC patients was a prognostic factor for poorer survival. Circulating DNA may be used as a non-invasive biomarker to refine the prognostic profile in NSCLC patients.

DNA microarray format for detection and subtyping of human papillomavirus.

A new human papillomavirus (HPV) assay using high-density DNA microarrays is described. An HPV DNA fragment from the 3' end of the E1 gene was amplified and digoxigenin labeled by PCR, and the resulting amplicons were hybridized onto type-specific oligonucleotides immobilized on high-density DNA microarrays. For detection, a simple immunohistochemical staining procedure was used with a substrate that has both colorimetric and fluorescent properties. This detection chemistry enables the rapid identification of reactive spots by regular light microscopy and semiquantification by laser scanning. Both single and multiple HPV infections are recognized by this assay, and the corresponding HPV types are easily identified. With this assay, 53 mucosal HPV types were detected and identified. A total of 45 HPV types were identified by a single type-specific probe, whereas the remaining 8 mucosal HPV types could be identified by a specific combination of probes. The simple assay format allows usage of this assay without expensive equipment, making it accessible to all diagnostic laboratories with PCR facilities.