Karl Vandepoele

The NBPF1 Promoter Has Been Recruited from the Unrelated EVI5 Gene Before Simian Radiation

Most new genes arise through the duplication of existing genes. In most cases, the duplication is not limited to the coding sequence but encompasses the regulatory region as well. The NBPF gene family has expanded during recent primate evolution, and it has no known mouse ortholog. One of its members, NBPF1, was found to be disrupted by a constitutional translocation in a neuroblastoma patient. Here, we show that the ancestral NBPF gene copied the regulatory region from an unrelated gene, EVI5, after the split between simians and prosimians but before simian radiation. Phylogenetic analysis points to the possible involvement of positive selection acting on the NBPF1 promoter in the simian lineage. We previously showed decreased NBPF1 expression in certain neuroblastoma cell lines. Here, we show that this expression pattern is mimicked by the EVI5 gene, but partly by different mechanisms. Epigenetic regulation of the EVI5 promoter is common in neuroblastoma cell lines, but it is not for the NBPF promoters. Here, we describe the recent acquisition of the NBPF1 promoter from an unrelated gene, and remarkably, both the donor (EVI5) and acceptor (NBPF1) genes are disrupted by constitutional translocations in patients with neuroblastoma, suggesting a functional link between these genes and the disease.

The association between fructosamine-3 kinase 900C/G polymorphism, transferrin polymorphism and human herpesvirus-8 infection in diabetics living in South Kivu.

Prevalences of human herpesvirus-8 (HHV-8) infection and diabetes mellitus are very common in certain parts of Africa, containing iron-rich soils. We hypothesized that some genetic factors could have a link with susceptibility to HHV-8 infection. We focused on ferroportin Q248H mutation (rs11568350), transferrin (TF) polymorphism and fructosamine-3 kinase (FN3K) 900C/G polymorphism (rs1056534). The study population consisted of 210 type 2 diabetic adults and 125 healthy controls recruited in Bukavu (South Kivu). In the whole study population (diabetics+healthy controls), ferroportin Q248H mutation was detected in 47 subjects (14.0%) with 43 heterozygotes and 4 homozygotes. TF phenotype frequencies were 88.1% (CC), 10.4% (CD) and 1.5% (BC). Genotype frequencies of FN3K 900C/G polymorphism were respectively 9,3% (CC), 43.3% (GC) and 47.4% (GG). Prevalence of HHV8-infection in the study population was 77.3%. HHV-8 infection rate and HHV-8 IgG antibody titer were significantly higher in diabetics then in controls (p<0.0001). Significant differences were observed in HHV-8 infection rate and in HHV-8 IgG antibody titer according to FN3K rs1056534 (p<0.05 and p<0.05, respectively) and TF polymorphism (p<0.05 and p=0.005, respectively). No significant differences in HHV-8 infection rate and in HHV-8 IgG antibody titer were observed in the ferroportin Q248H mutation carriers (rs11568350) in comparison with ferroportin wild type. In a multiple regression analysis, FN3K rs1056534, TF polymorphism and presence of diabetes mellitus were predictors for HHV-8 infection. In contrast to these findings, ferroportin Q248H mutation (rs11568350) did not influence the susceptibility for an HHV-8 infection in sub-Saharan Africans.

Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma

Background The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. Methods We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. Results PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. Conclusions In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair.

The YPEL5-PPP1CB fusion transcript is detected in different hematological malignancies and in normal samples.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in Western adults. It was suggested that transcripts from a reciprocal trans-splicing event between YPEL5 and PPP1CB were present exclusively in CLL patients (more than 90%). Here we show that the YPEL5–PPP1CB fusion is not specific for CLL but is also detected in other hematological malignancies such as chronic myeloid leukemia, monoclonal B cell lymphocytosis or acute leukemia and also in normal samples. As such, it is unlikely that the YPEL5–PPP1CB fusion is a good drug target in CLL or a suitable target to monitor disease.

The NBPF Gene Family

Neuroblastoma is one of the most intensely studied solid malignancies that affect children (Maris & Matthay, 1999). These tumours are heterogeneous biologically and clinically. One subset of neuroblastoma is susceptible to spontaneous apoptosis with little or no therapy and another subset differentiates over time, but most of these tumours are difficult to cure with current treatments. A relatively high proportion of affected children die due to resistance of the neuroblastoma to therapy (Van Roy et al., 2009). Every step in the identification or functional understanding of the genes that play important roles in this cancer could bring us closer to understanding the molecular mechanism and could help to develop more effective therapy. We will describe the novel NBPF gene family and its role in neuroblastoma. The NBPF gene family was originally identified by the disruption of one of its members in a neuroblastoma patient (Vandepoele et al., 2008). This gene was named NBPF1, for Neuroblastoma Breakpoint Family, member 1. Several reports indicate that the NBPF gene family might play an important role in neuroblastoma and possibly in other cancers as well. Additionally, this evolutionarily recent gene family has been involved as an important player in human evolution.

Pinpointing a potential role for CLEC12B in cancer predisposition through familial exome sequencing

Predisposition to cancer is only partly understood, and thus, the contribution of still undiscovered cancer predisposing variants necessitates further research. In search of such variants, we performed exome sequencing on the germline DNA of a family with two children affected by ganglioneuroma and neuroblastoma. Applying stringent selection criteria, we identified a potential deleterious, missense mutation in CLEC12B, coding for a lectin C‐type receptor that is predicted to regulate immune function. Although further screening in a larger population and functional characterization is needed, we propose CLEC12B as a candidate cancer predisposition gene.

A case of chronic eosinophilic leukemia with secondary transformation to acute myeloid leukemia

The natural history of primary eosinophilia remains highly variable and is characterized by underlying disease heterogeneity. Chronic eosinophilic leukemia, not otherwise specified (CEL-NOS) is a rare and aggressive disease characterized by non-specific cytogenetic abnormalities or elevated blasts, with high risk of transformation to acute leukemia. We describe a case of CEL-NOS with two hierarchically related non-specific cytogenetic rearrangements, associated with an NPM1 mutation and followed by evolution to secondary AML. NPM1 mutations are not previously described in CEL-NOS.

The NF1 hotspot in acute myeloid leukemia: what’s in a name?

Eisfeld and colleagues [1] recently described recurrent mutations in NF1 in patients with acute myeloid leukemia (AML), with a higher frequency than seen in previous studies. They show that mutations in hotspot Thr676 are associated with lower complete remission rates and shorter overall survival in patients <60 years. One of the factors that may have contributed to the previous oversight of this hotspot in previous studies, is its location in a mononucleotide cytosine repeat, which can easily be missed due to a faulty interpretation of sequencing data. Changes in mononucleotide repeats are cumbersome in their technical analysis but also in their nomenclature. Initiatives by the Human Genome Variation Society (HGVS) are trying to limit the nomenclature variability by providing clear guidelines [2] how to describe such variants. The authors refer to the NF1 hotspot as “p.Thr676fs*24 [c.2026dupC], Thr676”. This annotation is in conflict with the HGVS guidelines in several ways: as the DNA sequence is determined, this should be prominent, and the derived amino acid sequence should be between brackets (as it is extrapolated from the change at the DNA level). Applying this rule, the annotation would change to “c.2026dupC; p. (Thr676fs*24)”. However, no reference sequence is provided, and nucleotide 2026 is not a C in the two reference sequences (NM_000267.3 and NM_001042492.2) used to determine the Locus Reference Genomic transcripts [3]. In both transcripts, the C-stretch runs from nucleotides 2027 to 2033. As stated in the HGVS guidelines [2], for duplications in stretches of repeated sequences, the most 3′ residue is arbitrarily assigned to have been changed, resulting in a reassignment of the NF1 hotspot to c.2033 in the DNA sequence and from Thr676 to Ile679 in the protein. In conclusion, the HGVS compliant nomenclature of an additional cytosine in this C-stretch is NM_001042492.2: c.2033dup; p.(Ile679Aspfs*21). The Ile679 amino acid annotation is also used in the AACR GENIE dataset, to which the authors refer to confirm their results. The recognition of Ile679 as a hotspot for NF1 mutations, if confirmed in other studies, would benefit significantly from the use of a uniform nomenclature, following the HGVS guidelines.

Cancer-related mRNA expression analysis using a novel flow cytometry-based assay

Cancer‐related gene expression data mostly originate from unfractionated bulk samples, leading to “expression averaging” of heterogeneous populations. Multicolor flow cytometry (FCM) may distinguish heterogeneous populations based on the phenotypic characterization of single‐cells, but is not applicable for RNA targets. Here, we evaluated the PrimeFlow™ RNA assay, a novel FCM‐based assay designed to measure gene expressions, in two cancer entities with high and low RNA target levels.

RNA-based FLT3-ITD allelic ratio is associated with outcome and ex vivo response to FLT3 inhibitors in pediatric AML

Controversy exists whether internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-internal tandem duplication [ITD]) allelic ratio (AR) and/or length of the ITD should be taken into account for risk stratification of pediatric acute myeloid leukemia (AML) and whether it should be measured on RNA or DNA. Moreover, the ITD status may be of relevance for selecting patients eligible for FLT3 inhibitors. Here, we included 172 pediatric AML patients, of whom 36 (21%) harbored FLT3-ITD as determined on both RNA and DNA. Although there was a good correlation between both parameters ARspearman = 0.62 (95% confidence interval, 0.22-0.87) and ITDlengthspearman = 0.98 (95% confidence interval, 0.90-1.00), only AR ≥ 0.5 and length ≥48 base pairs (bps) based on RNA measurements were significantly associated with overall survival (AR: Plogrank = .008; ITDlength: Plogrank = .011). In large ITDs (>156 bp on DNA) a remarkable 90-bp difference exists between DNA and RNA, including intron 14, which is spliced out in RNA. Ex vivo exposure (n = 30) to FLT3 inhibitors, in particular to the FLT3-specific inhibitor gilteritinib, showed that colony-forming capacity was significantly more reduced in FLT3-ITD-AR ≥ 0.5 compared with ITD-AR-low and ITD- patient samples (P < .001). RNA-based FLT3-ITD measurements are recommended for risk stratification, and the relevance of AR regarding eligibility for FLT3-targeted therapy warrants further study.