The presence of germline mutations affecting the MYC-associated protein X (
MAX
) gene has recently been identified as one of the now 11 major genetic predisposition factors for the development of ...hereditary pheochromocytoma and/or paraganglioma. Little is known regarding how missense variants of unknown significance (VUS) in
MAX
affect its pivotal role in the regulation of the MYC/MAX/MXD axis. In the present study, we propose a consensus computational prediction based on five “state-of-the-art” algorithms. We also describe a PC12-based functional assay to assess the effects that 12
MAX
VUS may have on MYC’s E-box transcriptional activation. For all but two of these 12 VUS, the functional assay and the consensus computational prediction gave consistent results; we classified seven variants as pathogenic and three as nonpathogenic. The introduction of wild-type
MAX
cDNA into PC12 cells significantly decreased MYC’s ability to bind to canonical E-boxes, while pathogenic MAX proteins were not able to fully repress MYC activity. Further clinical and molecular evaluation of variant carriers corroborated the results obtained with our functional assessment. In the absence of clear heritability, clinical information, and molecular data, consensus computational predictions and functional models are able to correctly classify VUS affecting
MAX
.
Key messages
A functional assay assesses the effects of MAX VUS over MYC transcriptional activity.
A consensus computational prediction and the functional assay show high concordance.
Variant carriers’ clinical and molecular data support the functional assessment.
The aim of this study is to assess if common genetic variants located in the CDKN1B locus, coding for the cell cycle inhibitor p27Kip1, are involved in thyroid cancer susceptibility. Based on the ...literature and functional predictions, we selected three polymorphisms within the CDKN1B gene (rs2066827 (T326G, V109G), rs34330 (−79C>T) and rs36228499 (−838C>A)) to perform the first case–control study in thyroid cancer involving this locus. We had 649 Spanish patients with sporadic thyroid cancer and 385 healthy representative controls available. Luciferase reporter gene assays, real-time quantitative reverse transcription-PCR and immunoblot experiments were carried out to demonstrate the putative effect of the associated variant. The polymorphism rs34330 (−79C>T) was identified as a risk factor for developing the follicular variant of papillary thyroid carcinoma (FVPTC), fitting a recessive model (odds ratio=2.12; 95% confidence interval=1.09–4.15; P value=0.023). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27Kip1 promoter (P value <0.001). This effect was observed in −79TT genotype control carriers, who showed a tendency towards lower CDKN1B mRNA levels in lymphocytes, as well as at the protein level. This is the first study that identifies CDKN1B as a low-penetrance gene in thyroid cancer, and specifically in FVPTC subtype. We propose a reduced CDKN1B gene transcription depending on the genotype of the −79C>T (rs34330) variant as a novel mechanism underlying p27Kip1 downregulation.
Medullary thyroid carcinoma accounts for 2% to 5% of thyroid malignancies, of which 75% are sporadic and the remaining 25% are hereditary and related to multiple endocrine neoplasia type 2 syndrome. ...Despite a genotype-phenotype correlation with specific germline RET mutations, knowledge of pathways specifically associated with each mutation and with non- RET -mutated sporadic MTC remains lacking. Gene expression patterns have provided a tool for identifying molecular events related to specific tumor types and to different clinical features that could help identify novel therapeutic targets. Using transcriptional profiling of 49 frozen MTC specimens classified as RET mutation, we identified PROM1 , LOXL2 , GFRA1 , and DKK4 as related to RET M918T and GAL as related to RET 634 mutation. An independent series of 19 frozen and 23 formalin-fixed, paraffin-embedded (FFPE) MTCs was used for validation by RT-qPCR. Two tissue microarrays containing 69 MTCs were available for IHC assays. According to pathway enrichment analysis and gene ontology biological processes, genes associated with the MTCM918T group were involved mainly in proliferative, cell adhesion, and general malignant metastatic effects and with Wnt, Notch, NFκB, JAK/Stat, and MAPK signaling pathways. Assays based on silencing of PROM1 by siRNAs performed in the MZ-CRC-1 cell line, harboring RET M918T , caused an increase in apoptotic nuclei, suggesting that PROM1 is necessary for survival of these cells. This is the first report of PROM1 overexpression among primary tumors.
Pheochromocytomas (PCCs) and paragangliomas (PGLs) are tumors arising from the adrenal medulla and sympathetic/parasympathetic paraganglia, respectively. Approximately 40% of PCCs/PGLs are due to ...germline mutations in one of 16 susceptibility genes, and a further 30% are due to somatic alterations in 5 main genes. Recently, somatic ATRX mutations have been found in succinate dehydrogenase (SDH)-associated hereditary PCCs/PGLs. In the present study we applied whole-exome sequencing to the germline and tumor DNA of a patient with metastatic composite PCC and no alterations in known PCC/PGL susceptibility genes. A somatic loss-of-function mutation affecting ATRX was identified in tumor DNA. Transcriptional profiling analysis classified the tumor within cluster 2 of PCCs/PGLs (without SDH gene mutations) and identified downregulation of genes involved in neuronal development and homeostasis ( NLGN4 , CD99 and CSF2RA ) as well as upregulation of Drosha , an important gene involved in miRNA and rRNA processing. CpG island methylator phenotype typical of SDH gene-mutated tumors was ruled out, and SNP array data revealed a unique profile of gains and losses. Finally, we demonstrated the presence of alternative lengthening of telomeres in the tumor, probably associated with the failure of ATRX functions. In conclusion, somatic variants affecting ATRX may play a driver role in sporadic PCC/PGL.
Tyrosine kinase inhibitors (TKIs) have achieved remarkable clinical results in medullary thyroid carcinoma (MTC) patients. However, the considerable variability in patient response to treatment with ...TKIs remains largely unexplained. There is evidence that it could be due, at least in part, to alterations in genes associated with the disease via their effect on the expression of TKI targets. The objective of this study was to evaluate the influence of RAS mutations on the expression levels in MTC tumors of eight key TKI target proteins.
We assessed by immunohistochemistry the expression of EGFR, KIT, MET, PDGFRB, VEGF, VEGFR1, VEGFR2, and VEGFR3 in a series of 84 primary MTC tumors that had previously been molecularly characterized, including 14 RAS-positive, 18 RET(M918T)-positive, and 24 RET(C634)-positive tumors, as well as 15 wild-type tumors with no mutations in the RET or RAS genes.
In contrast to RET-positive tumors, RAS-positive tumors expressed neither PDGFRB nor MET (p=0.0060 and 0.047, respectively). Similarly, fewer RAS-positive than RET-related tumors expressed VEGFR3 (p=0.00062). Finally, wild-type tumors expressed VEGF more often than both RAS- and RET-positive tumors (p=0.0082 and 0.011, respectively).
This is the first study identifying that the expression of TKI targets differs according to the presence of RAS mutations in MTC. This information could potentially be used to select the most beneficial TKI treatment for these patients.
Cellular microtubules composed of α-β-tubulin heterodimers that are essential for cell shape, division, and intracellular transport are valid targets for anticancer therapy. However, not all the ...conserved but differentially expressed members of the β-tubulin gene superfamily have been investigated for their role in these settings. In this study, we examined roles for the hematologic isoform β-tubulin VI and functional genetic variants in the gene. β-tubulin VI was highly expressed in blood cells with a substantial interindividual variability (seven-fold variation in mRNA). We characterized DNA missense variations leading to Q43P, T274M, and R307H, and a rare nonsense variant, Y55X. Because variations in the hematologic target of microtubule-binding drugs might alter their myelosuppressive action, we tested their effect in cell lines stably expressing the different β-tubulin VI full-length variants, finding that the T274M change significantly decreased sensitivity to paclitaxel-induced tubulin polymerization. Furthermore, patients treated with paclitaxel and carrying β-tubulin VI T274M exhibited a significantly lower thrombocytopenia than wild-type homozygous patients (P = 0.031). Together, our findings define β-tubulin VI as a hematologic isotype with significant genetic variation in humans that may affect the myelosuppresive action of microtubule-binding drugs. A polymorphism found in a tubulin isoform expressed only in hemapoietic cells may contribute to the patient variation in myelosuppression that occurs after treatment with microtubule-binding drugs.
The therapeutic options for patients with metastatic medullary thyroid carcinoma (MTC) have recently increased due to the development of tyrosine kinase inhibitors (TKIs), some of which have achieved ...remarkable clinical responses in MTC patients. However, the molecular basis for the large variability in TKI responses is unknown. In this exploratory study, we investigated the expression of eight key TKI target proteins (EGFR, KIT, MET, PDGFRB, VEGF (VEGFA), VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4)) by immunohistochemistry in 103 molecularly characterized MTC samples and identified the associated clinical and molecular features. A number of MTC samples exhibited a high expression of VEGFR2 and VEGFR3, which were overexpressed in 57 and 43% of the MTC samples respectively. VEGFR1, PDGFRB, VEGF, KIT, and MET were present in 34–20% of the cases, while EGFR was highly expressed in only 10% of the MTC samples. Some proteins exhibited large differences in expression between sporadic and familial cases, suggesting that different RET mutations may be associated with the immunohistochemical profiles. MTC samples with the C634 RET mutation exhibited a higher expression of VEGFR3 and KIT than the M918T RET-mutated and non-mutated RET tumor samples (P=0.005 and P=0.007 respectively) and a lower expression of VEGFR1 (P=0.04). Non-mutated RET MTC cases exhibited a lower expression of PDGFRB (P=0.04). Overall, this is the first study, to our knowledge, to show that multiple TKI targets are highly expressed in a subset of MTCs, suggesting that molecular stratification of patients may have the potential to improve TKI therapies for MTC.
Cytochrome P450 2D6 (CYP2D6) copy number variation (CNV) influences the metabolism of 15–25% of clinical drugs. Here we describe a novel multiplex polymerase chain reaction (PCR) analysis method that ...accurately detects
CYP2D6 CNV and
CYP2D6*9 allele. It includes the amplification of 2
CYP2D6 and 7 control (
AQP1, CYP3A4, MDR1, and
SDHB) fluorescent PCR products that are separated on a capillary sequencer and normalized using reference samples. The technique was validated using 27 PCR–restriction fragment length polymorphism (RFLP) pregenotyped samples and further tested in 75 Caucasian samples. The method assigns the correct
CYP2D6 copy number, independent of already characterized
CYP2D6 single nucleotide polymorphisms (SNPs), and could easily be applied to clinical samples.
To date, few association studies have been done to better understand the genetic basis for the development of sporadic medullary thyroid carcinoma (sMTC). To identify additional low-penetrance genes, ...we have done a two-stage case-control study in two European populations using high-throughput genotyping. We selected 417 single nucleotide polymorphisms (SNP) belonging to 69 genes either related to RET signaling pathway/functions or involved in key processes for cancer development. TagSNPs and functional variants were included where possible. These SNPs were initially studied in the largest known series of sMTC cases (n = 266) and controls (n = 422), all of Spanish origin. In stage II, an independent British series of 155 sMTC patients and 531 controls was included to validate the previous results. Associations were assessed by an exhaustive analysis of individual SNPs but also considering gene- and linkage disequilibrium-based haplotypes. This strategy allowed us to identify seven low-penetrance genes, six of them (STAT1, AURKA, BCL2, CDKN2B, CDK6, and COMT) consistently associated with sMTC risk in the two case-control series and a seventh (HRAS) with individual SNPs and haplotypes associated with sMTC in the Spanish data set. The potential role of CDKN2B was confirmed by a functional assay showing a role of a SNP (rs7044859) in the promoter region in altering the binding of the transcription factor HNF1. These results highlight the utility of association studies using homogeneous series of cases for better understanding complex diseases.
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