[Show abstract][Hide abstract] ABSTRACT: Heimler syndrome (HS) is a rare recessive disorder characterized by sensorineural hearing loss (SNHL), amelogenesis imperfecta, nail abnormalities, and occasional or late-onset retinal pigmentation. We ascertained eight families affected by HS and, by using a whole-exome sequencing approach, identified biallelic mutations in PEX1 or PEX6 in six of them. Loss-of-function mutations in both genes are known causes of a spectrum of autosomal-recessive peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome. PBDs are characterized by leukodystrophy, hypotonia, SNHL, retinopathy, and skeletal, craniofacial, and liver abnormalities. We demonstrate that each HS-affected family has at least one hypomorphic allele that results in extremely mild peroxisomal dysfunction. Although individuals with HS share some subtle clinical features found in PBDs, the diagnosis was not suggested by routine blood and skin fibroblast analyses used to detect PBDs. In conclusion, our findings define HS as a mild PBD, expanding the pleiotropy of mutations in PEX1 and PEX6.
Full-text · Article · Sep 2015 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: The advent of anti-EGFR (epidermal growth factor receptor) therapy resulted in significant progress in the treatment of metastatic colorectal cancer patients. However, many patients do not respond to this therapy or develop acquired resistance within a few months after the start of treatment. Since 2008, anti-EGFR therapy is restricted to KRAS wild-type patients as it has been shown that KRAS exon 2-mutated patients do not respond to this therapy. Still, up to 60 % of KRAS exon 2 wild-type patients show primary resistance to this treatment. Recently, several studies investigating the predictive and prognostic role of RAS mutations other than in KRAS exon 2 demonstrated that patients with these mutations are not responding to therapy. However, the role of these mutations has long been questioned as The National Comprehensive Cancer Network Guidelines in Oncology and the European Medicines Agency indications had already been changed in order to restrict anti-EGFR therapy to all RAS wild-type colorectal cancer patients, while the Food and Drug Administration guidelines remained unchanged. Recently, the Food and Drug Administration guidelines have also been changed, which implies the importance of RAS mutations beyond KRAS exon 2 in colorectal cancer. In this review, we discuss the most important studies regarding the predictive and prognostic role of RAS mutations other than in KRAS exon 2 in order to demonstrate the importance of these RAS mutations in patients with metastatic colorectal cancer treated with anti-EGFR therapy.
[Show abstract][Hide abstract] ABSTRACT: After an initial response to EGFR targeted therapy, secondary resistance almost invariably ensues, thereby limiting the clinical benefit of the drug. Hence, it has been recognized that the successful implementation of targeted therapy in the treatment of HNSCC cancer is very much dependent on predictive biomarkers for patient selection.
We generated an in vitro model of acquired cetuximab resistance by chronically exposing three HNSCC cell lines to increasing cetuximab doses. Gene expression profiles of sensitive parental cells and resistant daughter cells were compared using microarray analysis. Growth inhibitory experiments were performed with an HB-EGF antibody and the MMP inhibitor, both in combination with cetuximab. Characteristics of EMT were analyzed using migration and invasion assays, immunofluorescent vimentin staining and qRT-PCR for several genes involved in this process. The function of the transcription factor AP-1 was investigated using qRT-PCR for several genes upregulated or downregulated in cetuximab resistant cells. Furthermore, anchorage-independent growth was investigated using the soft agar assay.
Gene expression profiling shows that cetuximab resistant cells upregulate several genes, including interleukin 8, the EGFR ligand HB-EGF and the metalloproteinase ADAM19. Cytotoxicity experiments with neutralizing HB-EGF antibody could not induce any growth inhibition, whereas an MMP inhibitor inhibited cell growth in cetuximab resistant cells. However, no synergetic effects combined with cetuximab could be observed. Cetuximab resistant cells showed traits of EMT, as witnessed by increased migratory potential, increased invasive potential, increased vimentine expression and increased expression of several genes involved in EMT. Furthermore, expression of upregulated genes could be repressed by the treatment with apigenin. The cetuximab resistant LICR-HN2 R10.3 cells tend to behave differently in cell culture, forming spheres. Therefore, soft agar assay was performed and showed more and larger colonies when challenged with cetuximab compared to PBS challenged cells.
In summary, our results indicate that increased expression of the ligand HB-EGF could contribute to resistance towards cetuximab in our cetuximab resistant HNSCC cells. Furthermore, several genes upregulated or downregulated in cetuximab resistant cells are under control of the AP-1 transcription factor. However, more studies are warranted to further unravel the role of AP-1 in cetuximab resistance.
No preview · Article · Aug 2015 · American Journal of Cancer Research
[Show abstract][Hide abstract] ABSTRACT: A 3-year-old female patient presenting with an unknown syndrome of a neonatal progeroid appearance, lipodystrophy, pulmonary hypertension, cutis marmorata, feeding disorder and failure to thrive was investigated by whole-genome sequencing. This revealed a de novo, heterozygous, frame-shift mutation in the Caveolin1 gene (CAV1) (p.Phe160X). Mutations in CAV1, encoding the main component of the caveolae in plasma membranes, cause Berardinelli-Seip congenital lipodystrophy type 3 (BSCL). Although BSCL is recessive, heterozygous carriers either show a reduced phenotype of partial lipodystrophy, pulmonary hypertension, or no phenotype. To investigate the pathogenic mechanisms underlying this syndrome in more depth, we performed next generation RNA sequencing of peripheral blood, which showed several dysregulated pathways in the patient that might be related to the phenotypic progeroid features (apoptosis, DNA repair/replication, mitochondrial). Secondly, we found a significant down-regulation of known Cav1 interaction partners, verifying the dysfunction of CAV1. Other known progeroid genes and lipodystrophy genes were also dysregulated. Next, western blotting of lysates of cultured fibroblasts showed that the patient shows a significantly decreased expression of wild-type CAV1 protein, demonstrating a loss-of-function mutation, though her phenotype is more severe that other heterozygotes with similar mutations. This phenotypic variety could be explained by differences in genetic background. Indications for this are supported by additional rare variants we found in AGPAT2 and LPIN1 lipodystrophy genes. CAV1, AGPAT2 and LPIN1 all play an important role in triacylglycerol (TAG) biosynthesis in adipose tissue, and the defective function in different parts of this pathway, though not all to the same extend, could contribute to a more severe lipoatrophic phenotype in this patient. In conclusion, we report, for the first time, an association of CAV1 dysfunction with a syndrome of severe premature aging and lipodystrophy. This may contribute to a better understanding of the aging process and pathogenic mechanisms that contribute to premature aging.
[Show abstract][Hide abstract] ABSTRACT: Cell death exists in many different forms. Some are accidental, but most of them have some kind of regulation and are called programmed cell death. Programmed cell death (PCD) is a very diverse and complex mechanism and must be tightly regulated. This study investigated PCD induced by DFNA5, a gene responsible for autosomal dominant hearing loss (HL) and a tumor suppressor gene (TSG) involved in frequent forms of cancer. Mutations in DFNA5 lead to exon 8 skipping and result in HL in several families. Expression of mutant DFNA5, a cDNA construct where exon 8 is deleted, was linked to PCD both in human cell lines and in Saccharomyces cerevisiae. To further investigate the cell death mechanism induced by mutant DFNA5, we performed a microarray study in both models. We used wild-type DFNA5, which does not induce cell death, as a reference. Our data showed that the yeast pathways related to mitochondrial ATP-coupled electron transport chain, oxidative phosphorylation and energy metabolism were up-regulated, while in human cell lines, MAP kinase-related activity was up-regulated. Inhibition of this pathway was able to partially attenuate the resulting cell death induced by mutant DFNA5 in human cell lines. In yeast, the association with mitochondria was demonstrated by up-regulation of several cytochrome c oxidase (COX) genes involved in the cellular oxidative stress production. Both models show a down-regulation of protein sorting- and folding-related mechanisms suggesting an additional role for the endoplasmic reticulum (ER). The exact relationship between ER and mitochondria in DFNA5-induced cell death remains unknown at this moment, but these results suggest a potential link between the two.
[Show abstract][Hide abstract] ABSTRACT: Introduction and aim Recently a new phenotypic and characteristic radiologic feature of DNA9 has been discovered in a series of 9 patients, all with the same PSIS COCH gene mutation. This study adds more patients to the series. Material and methods CT en MRI imaging of 14 patients who presented between 2007 and 2014 with otovestibular deterioration all caused by the same c.1S IC>T,p.ProS ISet (PSIS) missense mutation in the COCH gene, were analyzed. The mean patients' age at the time of investigation was 62 years, and 29% were female. Results Ninety-three percent of the patients had sclerotic lesions and/or narrowing in one or more semicircular canals on CT scan with a signal loss at corresponding areas on T2-weighted MR images. The posterior canals were affected in most cases (50%), compared with the superior (25%) and lateral canals (14%) or the vestibule (4%). Only 77% of the lesions on MR images were also visible on CT scans, suggesting a fibrotic process without calcification. As the disease progresses, patients get more lesions visible on imaging and more severe hearing loss. Median PTA in unaffected ears is 60 dB HL versus 100 dB HL in affected ears. Conclusions An extended retrospective analyses of CT and MRI imaging in patients with the same P51S mutation in the COCH gene, confirmed the recently discovered characteristic radiologic feature.
[Show abstract][Hide abstract] ABSTRACT: The human BON-1 and QGP-1 cell lines are two frequently used models in pancreatic neuroendocrine tumor (PNET) research. Data on the whole exome genetic constitution of these cell lines is largely lacking. This study presents the first whole exome profile of the BON-1 and QGP-1 cell lines. Cell line identity was confirmed by short tandem repeat profiling. Using GTG-banding and CytoSNP-12v2 Beadchip array, cell line ploidy and chromosomal alterations were determined in BON-1 and QGP-1. The exome of both cell lines was sequenced on Ilumina's HiSeq next generation sequencing (NGS) platform. Single Nucleotide Variants (SNVs) and indels were called using the Genome Analysis ToolKit. SNVs were validated with Sanger sequencing. Ploidy of BON-1 and QGP-1 was 3 and 4 respectively, with long stretches of loss of heterozygosity across multiple chromosomes, which is associated with aggressive tumor behaviour. In BON-1, 57 frameshift indels and 1725 possible protein-altering SNVs were called in the NGS data. In the QGP-1 cell line, 56 frameshift indels and 1095 SNVs were identified. ATRX, a PNET-associated gene, was mutated in both cell lines, while TSC2 contained a mutation in BON-1. NRAS showed a mutation in BON-1, while KRAS was mutated in QGP-1, implicating aberrations in the RAS pathway in both cell lines. Homozygous mutations in TP53 with possible loss of function were identified in both cell lines. Various MUC genes, implicated in cell signaling, lubrication and chemical barriers, which are frequently expressed in PNET tissue samples, showed homozygous protein-altering SNVs in the BON-1 and QGP-1 cell line.
No preview · Article · Jan 2015 · Journal of Molecular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC-inducible mir-150-5p adds another level of regulation to GC specific therapeutic responses in multiple myeloma.
[Show abstract][Hide abstract] ABSTRACT: Anti-EGFR therapy in metastatic colorectal cancer (mCRC) has improved survival outcome. However, many patients do not respond to this therapy and almost all patients develop resistance after a few months of treatment. Since 2008, the therapy has been restricted to patients without mutations in KRAS, an important target in the EGFR pathway, as these patients do not benefit from anti-EGFR therapy. Recently, this has been changed to an all-RAS wild-type strategy. Despite these restrictions, still 40 to 60 % of mCRC patients are resistant. New biomarkers need to be identified in order to improve patient selection. Another problem is tumor heterogeneity, which impedes the detection of mutations in resistance genes and can consequently lead to wrong treatment decisions. A possible solution for this problem may be found in liquid biopsies. In this review, known and promising upcoming biomarkers associated with resistance to anti-EGFR therapy will be summarized. Moreover, the potential added value of liquid biopsies in patient selection and follow-up will be discussed.
Full-text · Article · Dec 2014 · Current Colorectal Cancer Reports
[Show abstract][Hide abstract] ABSTRACT: In the present article, the utility of a special potentiometric titration approach for recognition and calculation of biomolecule/small molecule interactions is reported. This approach is fast, sensitive, reproducible and inexpensive in comparison to the other methods for the determination of the association constant values (Ka) and the interaction energies (ΔG). The potentiometric titration measurement is based on the use of a classical polymeric membrane indicator electrode in a solution of the small molecule ligand. The biomolecule is used as a titrant. The potential is measured versus a reference electrode and transformed to a concentration related signal over the entire concentration interval, also at low concentrations, where the mV (y-axis) versus logcanalyte (x-axis) potentiometric calibration curve is not linear. In the procedure, the Ka is calculated for the interaction of cocaine with a cocaine binding aptamer and with an anti-cocaine antibody. To study the selectivity and cross-reactivity, other oligonucleotides and aptamers are tested, as well as other small ligand molecules such as tetrakis (4-chlorophenyl)borate, metergoline, lidocaine, and bromhexine. The calculated Ka compared favorably to the value reported in the literature using SPR. The potentiometric titration approach called "Concentration related Response Potentiometry", is used to study molecular interaction for 7 macromolecular target molecules and 4 small molecule ligands.
Full-text · Article · Nov 2014 · Analytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: The MDM2 promoter SNP285C is located on the SNP309G allele. While SNP309G enhances Sp1 transcription factor binding and MDM2 transcription, SNP285C antagonizes Sp1 binding and reduces the risk of breast-, ovary- and endometrial cancer. Assessing SNP285 and 309 genotypes across 25 different ethnic populations (>10.000 individuals), the incidence of SNP285C was 6-8% across European populations except for Finns (1.2%) and Saami (0.3%). The incidence decreased towards the Middle-East and Eastern Russia, and SNP285C was absent among Han Chinese, Mongolians and African Americans. Interhaplotype variation analyses estimated SNP285C to have originated about 14,700 years ago (95% CI: 8,300 - 33,300). Both this estimate and the geographical distribution suggest SNP285C to have arisen after the separation between Caucasians and modern day East Asians (17,000 - 40,000 years ago). We observed a strong inverse correlation (r = -0.805; p < 0.001) between the percentage of SNP309G alleles harboring SNP285C and the MAF for SNP309G itself across different populations suggesting selection and environmental adaptation with respect to MDM2 expression in recent human evolution. In conclusion, we found SNP285C to be a pan-Caucasian variant. Ethnic variation regarding distribution of SNP285C needs to be taken into account when assessing the impact of MDM2 SNPs on cancer risk.
[Show abstract][Hide abstract] ABSTRACT: Unraveling the underlying mechanisms of cetuximab resistance in head and neck squamous cell carcinoma (HNSCC) is of major importance as many tumors remain non-responsive or become resistant. Our microarray results suggest that "resistant" cells still exhibit RAS-MAPK pathway signaling contributing to drug resistance, as witnessed by low expression of DUSP5 and DUSP6, negative regulators of ERK1/2, and increased expression of AURKB, a key regulator of mitosis. Therefore, interrupting the RAS-MAPK pathway by an ERK1/2 inhibitor (apigenin) or an AURKB inhibitor (barasertib) might be a new strategy for overcoming cetuximab resistance in HNSCC.
[Show abstract][Hide abstract] ABSTRACT: We performed a genome-wide association study (GWAS) to identify the genes responsible for age-related hearing impairment (ARHI), the most common form of hearing impairment in the elderly. Analysis of common variants, with and without adjustment for stratification and environmental covariates, rare variants and interactions, as well as gene-set enrichment analysis, showed no variants with genome-wide significance. No evidence for replication of any previously reported genes was found. A study of the genetic architecture indicates for the first time that ARHI is highly polygenic in nature, with probably no major genes involved. The phenotype depends on the aggregated effect of a large number of SNPs, of which the individual effects are undetectable in a modestly powered GWAS. We estimated that 22% of the variance in our data set can be explained by the collective effect of all genotyped SNPs. A score analysis showed a modest enrichment in causative SNPs among the SNPs with a P-value below 0.01.European Journal of Human Genetics advance online publication, 18 June 2014; doi:10.1038/ejhg.2014.56.
Full-text · Article · Jun 2014 · European journal of human genetics: EJHG