Nienke van der Stoep

Genome-wide association study identifies three new melanoma susceptibility loci

We report a genome-wide association study for melanoma that was conducted by the GenoMEL Consortium. Our discovery phase included 2,981 individuals with melanoma and 1,982 study-specific control individuals of European ancestry, as well as an additional 6,426 control subjects from French or British populations, all of whom were genotyped for 317,000 or 610,000 single-nucleotide polymorphisms (SNPs). Our analysis replicated previously known melanoma susceptibility loci. Seven new regions with at least one SNP with P < 10−5 and further local imputed or genotyped support were selected for replication using two other genome-wide studies (from Australia and Texas, USA). Additional replication came from case-control series from the UK and The Netherlands. Variants at three of the seven loci replicated at P < 10−3: an SNP in ATM (rs1801516, overall P = 3.4 × 10−9), an SNP in MX2 (rs45430, P = 2.9 × 10−9) and an SNP adjacent to CASP8 (rs13016963, P = 8.6 × 10−10). A fourth locus near CCND1 remains of potential interest, showing suggestive but inconclusive evidence of replication (rs1485993, overall P = 4.6 × 10−7 under a fixed-effects model and P = 1.2 × 10−3 under a random-effects model). These newly associated variants showed no association with nevus or pigmentation phenotypes in a large British case-control series.

Diagnostic guidelines for high-resolution melting curve (HRM) analysis: an interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner.

Genetic analysis of BRCA1 by sequencing is often preceded by a scanning method like denaturing gradient gel electrophoresis (DGGE), protein truncation test (PTT) or DHPLC. High‐resolution melting curve (HRM) analysis is a promising and economical method for high‐throughput mutation scanning. The EuroGentest network (www.eurogentest.org) aims to assist with the introduction of novel technologies in the diagnostic setting. Therefore, we have performed a thorough and high‐standard interlaboratory evaluation and validation of HRM, in collaboration with Idaho Technology, the manufacturer of the LightScannerTM (LS). Through this detailed study of 170 variants, we have generated guidelines for easy setup and implementation of HRM as a scanning technique for new genes, which are adaptable to the quality system of an individual diagnostic laboratory. This validation study includes the description of a BRCA1‐specific mutation screening test using the 96‐well LS. This assay comprises 40 amplicons and was evaluated using a statistically significant elaborate panel of variants and control DNA samples. All heterozygous variants were detected. Moreover, genotype analysis for nine common polymorphisms created a fast screening and detection method for these frequently occurring nonpathogenic variants. A blind study using a total of 28 patient‐derived DNA samples resulted also in 100% detection and showed an average specificity of 98%, indicating a low incidence of false positives (FPs). Hum Mutat 30:1–11, 2009.

Activated Human T Cells Accomplish MHC Class II Expression Through T Cell-Specific Occupation of Class II Transactivator Promoter III

Activated human T cells express HLA-DR, HLA-DQ, and HLA-DP on their surface, but the regulation and functioning of MHC class II molecules in T lymphocytes are poorly understood. Because the MHC class II transactivator (CIITA) is essential for MHC class II expression, we have investigated transcriptional activation of CIITA in activated T cells. In this study, we show that in human activated CD4+ T cells, CIITA promoter III (CIITA-PIII) drives the expression of CIITA. The in vivo genomic footprint analysis revealed activated T cell-specific occupation of CIITA-PIII. Subsequent EMSA analysis of several promoter regions showed differences in banding pattern among activated T cells, naive T cells, primary B cells, and Raji B cells. Activating response element (ARE)-1 is shown to interact with the acute myeloid leukemia 2 transcription factor in nuclear extracts derived from both T and B cells. Interestingly, the acute myeloid leukemia 3 transcription factor was bound in nuclear extracts of T cells only. The ARE-2 sequence is able to bind CREB/activating transcription factor family members in both T and B cells. In addition, a yet unidentified Ets family member was found to interact with site C in activated T cells, whereas in B cells site C was bound by PU.1 and Pip/IFN regulatory factor 4/IFN consensus sequence binding protein for activated T cells. In Jurkat T cells, both ARE-1 and ARE-2 are crucial for CIITA-PIII activity, similar to Raji B cells. The differential banding pattern in in vivo genomic footprinting and transcription factor binding at the ARE-1 and site C between T cells and B cells probably reflects differences in CIITA-PIII activation pathways employed by these cell types.

Genome-wide meta-analysis identifies five new susceptibility loci for cutaneous malignant melanoma

Thirteen common susceptibility loci have been reproducibly associated with cutaneous malignant melanoma (CMM). We report the results of an international 2-stage meta-analysis of CMM genome-wide association studies (GWAS). This meta-analysis combines 11 GWAS (5 previously unpublished) and a further three stage 2 data sets, totaling 15,990 CMM cases and 26,409 controls. Five loci not previously associated with CMM risk reached genome-wide significance (P < 5 × 10−8), as did 2 previously reported but unreplicated loci and all 13 established loci. Newly associated SNPs fall within putative melanocyte regulatory elements, and bioinformatic and expression quantitative trait locus (eQTL) data highlight candidate genes in the associated regions, including one involved in telomere biology.

Lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells and is caused by methylation of the IFN-γ inducible promoter (PIV) of CIITA

Lack of MHC-mediated antigen presenting functions of fetal trophoblast cells is an important mechanism to evade maternal immune recognition. In this study we demonstrated that the deficiency in MHC expression and antigen presentation in the trophoblast cell lines JEG-3 and JAR is caused by lack of class II transactivator (CIITA) expression due to hypermethylation of its interferon-gamma (IFN-gamma)-responsive promoter (PIV). Circumvention of this lack of CIITA expression by introduction of exogenous CIITA induced cell surface expression of HLA-DR, -DP, and -DQ, leading to an acquired capacity to present antigen to antigen-specific T cells. Transfection of CIITA in JEG-3 cells also upregulated functional HLA-B and HLA-C expression. Noteworthy, this lack of IFN-gamma-mediated induction of CIITA was also found to exist in normal trophoblast cells expanded from chorionic villus biopsies. Together, these observations demonstrate that lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells.

Best Practice Guidelines for the Use of Next-Generation Sequencing Applications in Genome Diagnostics: A National Collaborative Study of Dutch Genome Diagnostic Laboratories

Next‐generation sequencing (NGS) methods are being adopted by genome diagnostics laboratories worldwide. However, implementing NGS‐based tests according to diagnostic standards is a challenge for individual laboratories. To facilitate the implementation of NGS in Dutch laboratories, the Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL) set up a working group in 2012. The results of their discussions are presented here. We provide best practice guidelines and criteria for implementing and validating NGS applications in a clinical setting. We introduce the concept of “diagnostic yield” as the main performance characteristic for evaluating diagnostic tests. We recommend that the laboratory procedures, including the tested genes, should be recorded in a publicly available document describing the complete “diagnostic routing.” We also propose that laboratories should use a list of “core disease genes” for specific genetic diseases. This core list contains the essential genes for each disease, and they should all be included in a diagnostic test to establish a reliable and accurate molecular diagnosis. The guidelines will ensure a clear and standardized quality of care provided by genetic diagnostic laboratories. The best practice guidelines and criteria that are presented here were adopted by the VKGL in January 2013.

Lack of IFN-γ-mediated induction of the class II transactivator (CIITA) through promoter methylation is predominantly found in developmental tumor cell lines

Downregulation of major histocompatibility complex (MHC) molecules by tumor cells impairs cellular immune recognition and contributes to inefficient cell‐mediated tumor eradication. Low or lack of expression of MHC molecules is frequently observed in early developmental or embryonically derived tumor cells. Considering the central role of the class II transactivator (CIITA) in MHC class II‐ and class I‐mediated antigen presentation, we compared the induction of CIITA by interferon‐γ (IFN‐γ) in a diverse panel of developmental and more differentiated tumor cell lines. In contrast to the more differentiated tumor cell lines, none of the developmental tumor cell lines were capable of expressing CIITA after treatment with IFN‐γ. Remarkably, in transient transfection assays, CIITA promoter IV (CIITA‐PIV) was found to be induced by IFN‐γ. Southern blot analysis of genomic DNA obtained from the developmental tumor cell lines indicated that the absence of endogenous CIITA induction was due to methylation of the CIITA‐PIV region. Exposure to 5‐azacytidine restored induction of CIITA and congruent HLA‐DRA expression in these cells. The observation that only developmental tumor cell lines, originating from various tissues, employ methylation to silence CIITA expression may reflect the natural status of CIITA expression during early development rather than oncogenic transformation.

A variant in FTO shows association with melanoma risk not due to BMI

We report the results of an association study of melanoma that is based on the genome-wide imputation of the genotypes of 1,353 cases and 3,566 controls of European origin conducted by the GenoMEL consortium. This revealed an association between several SNPs in intron 8 of the FTO gene, including rs16953002, which replicated using 12,313 cases and 55,667 controls of European ancestry from Europe, the USA and Australia (combined P = 3.6 × 10−12, per-allele odds ratio for allele A = 1.16). In addition to identifying a new melanoma-susceptibility locus, this is to our knowledge the first study to identify and replicate an association with SNPs in FTO not related to body mass index (BMI). These SNPs are not in intron 1 (the BMI-related region) and exhibit no association with BMI. This suggests FTOs function may be broader than the existing paradigm that FTO variants influence multiple traits only through their associations with BMI and obesity.

DNA methylation and expression of major histocompatibility complex class I and class II transactivator genes in human developmental tumor cells and in T cell malignancies

Major histocompatibility complex (MHC) class I and class II molecules play essential roles in the immune response by virtue of their ability to present peptides to T lymphocytes. Given their central role in adaptive immunity, the genes encoding these peptide-presenting molecules are regulated in a tight fashion to meet with local requirements for an adequate immune response. In contrast to MHC class I gene products, which are expressed on almost all nucleated cells, constitutive expression of MHC class II molecules is found in specialized antigen presenting cells of the immune system only. Transcription of both MHC class I and class II genes can be induced by immune regulators and upon cell activation. Transcription of MHC class I genes is mediated by a set of conserved cis acting regulatory elements in their promoters. Of these regulatory elements, MHC class II promoters share the SXY-module. Essential for activation of MHC class II promoters is the class II transactivator (CIITA), which acts through protein/protein interactions with regulatory factors bound to the SXY module. In this review, we discuss the role of DNA methylation in relation to altered expression of MHC class I and CIITA genes as observed in malignancies and in development.

Next-generation sequencing-based genome diagnostics across clinical genetics centers: implementation choices and their effects

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found ‘no causal variant’, thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of ‘greediness’ to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care.