Marta J. Llorca-Cardeñosa

Long telomere length and a TERT-CLPTM1 locus polymorphism association with melanoma risk.

Telomere length has been associated with the development of cancer. Studies have shown that shorter telomere length may be related to a decreased risk of cutaneous melanoma. Furthermore, deregulation of the telomere-maintaining gene complexes, has been related to this oncogenic process. Some variants in these genes seem to be correlated with a change in telomerase expression. We examined the effect of 10 single nucleotide polymorphisms (SNPs) in the TERT gene (encoding telomerase), one SNP in the related TERT-CLPTM1L locus and one SNP in the TRF1 gene with telomere length, and its influence on melanoma risk in 970 Spanish cases and 733 Spanish controls. Genotypes were determined using KASP technology, and telomere length was measured by quantitative polymerase chain reaction (PCR) on DNA extracted from peripheral blood leucocytes. Our results demonstrate that shorter telomere length is associated with a decreased risk of melanoma in our population (global p-value, 2.69×10(-11)), which may be caused by a diminution of proliferative potential of nevi (melanoma precursor cells). We also obtained significant results when we tested the association between rs401681 variant (TERT-CLPTM1L locus) with melanoma risk (Odds ratio, OR; 95% confidence interval, CI=1.24 (1.08-1.43); p-value, 3×10(-3)). This is the largest telomere-related study undertaken in a Spanish population to date. Furthermore, this study represents a comprehensive analysis of some of the most relevant telomere pathway genes in relation to cutaneous melanoma susceptibility.

Deregulation of ARID1A, CDH1, cMET and PIK3CA and target-related microRNA expression in gastric cancer

Genetic and epigenetic alterations play an important role in gastric cancer (GC) pathogenesis. Aberrations of the phosphatidylinositol-3-kinase signaling pathway are well described. However, emerging genes have been described such as, the chromatin remodeling gene ARID1A. Our aim was to determine the expression levels of four GC-related genes, ARID1A, CDH1, cMET and PIK3CA, and 14 target-related microRNAs (miRNAs). We compared mRNA and miRNA expression levels among 66 gastric tumor and normal adjacent mucosa samples using quantitative real-time reverse transcription PCR. Moreover, ARID1A, cMET and PIK3CA protein levels were assessed by immunohistochemistry (IHC). Finally, gene and miRNAs associations with clinical characteristics and outcome were also evaluated. An increased cMET and PIK3CA mRNA expression was found in 78.0% (P = 2.20 × 10−5) and 73.8% (P = 1.00 × 10−3) of the tumors, respectively. Moreover, IHC revealed that cMET and PIK3CA expression was positive in 63.6% and 87.8% of the tumors, respectively. Six miRNAs had significantly different expression between paired-samples, finding five up-regulated [miR-223-3p (P = 1.65 × 10−6), miR-19a-3p (P = 1.23 × 10−4), miR-128-3p (P = 3.49 × 10−4), miR-130b-3p (P = 1.00 × 10−3) and miR-34a-5p (P = 4.00 × 10−3)] and one down-regulated [miR-124-3p (P = 0.03)]. Our data suggest that cMET, PIK3CA and target-related miRNAs play an important role in GC and may serve as potential targets for therapy.

Sex-specific genetic effects associated with pigmentation, sensitivity to sunlight, and melanoma in a population of Spanish origin

BackgroundHuman pigmentation is a polygenic quantitative trait with high heritability. In addition to genetic factors, it has been shown that pigmentation can be modulated by oestrogens and androgens via up- or down-regulation of melanin synthesis. Our aim was to identify possible sex differences in pigmentation phenotype as well as in melanoma association in a melanoma case-control population of Spanish origin.MethodsFive hundred and ninety-nine females (316 melanoma cases and 283 controls) and 458 males (234 melanoma cases and 224 controls) were analysed. We genotyped 363 polymorphisms (single nucleotide polymorphisms (SNPs)) from 65 pigmentation gene regions.ResultsWhen samples were stratified by sex, we observed more SNPs associated with dark pigmentation and good sun tolerance in females than in males (107 versus 75; P = 2.32 × 10−6), who were instead associated with light pigmentation and poor sun tolerance. Furthermore, six SNPs in TYR, SILV/CDK2, GPR143, and F2RL1 showed strong differences in melanoma risk by sex (P < 0.01).ConclusionsWe demonstrate that these genetic variants are important for pigmentation as well as for melanoma risk, and also provide suggestive evidence for potential differences in genetic effects by sex.

Determination of somatic oncogenic mutations linked to target-based therapies using MassARRAY technology

Somatic mutation analysis represents a useful tool in selecting personalized therapy. The aim of our study was to determine the presence of common genetic events affecting actionable oncogenes using a MassARRAY technology in patients with advanced solid tumors who were potential candidates for target-based therapies. The analysis of 238 mutations across 19 oncogenes was performed in 197 formalin-fixed paraffin-embedded samples of different tumors using the OncoCarta Panel v1.0 (Sequenom Hamburg, Germany). Of the 197 specimens, 97 (49.2%) presented at least one mutation. Forty-nine different oncogenic mutations in 16 genes were detected. Mutations in KRAS and PIK3CA were detected in 40/97 (41.2%) and 30/97 (30.9%) patients respectively. Thirty-one patients (32.0%) had mutations in two genes, 20 of them (64.5%) initially diagnosed with colorectal cancer. The co-occurrence of mutation involved mainly KRAS, PIK3CA, KIT and RET. Mutation profiles were validated using a customized panel and the Junior Next-Generation Sequencing technology (GS-Junior 454, Roche). Twenty-eight patients participated in early clinical trials or received specific treatments according to the molecular characterization (28.0%). MassARRAY technology is a rapid and effective method for identifying key cancer-driving mutations across a large number of samples, which allows for a more appropriate selection for personalized therapies.

Epigenetic changes in localized gastric cancer: the role of RUNX3 in tumor progression and the immune microenvironment.

Gastric cancer (GC) pathogenesis involves genetic, epigenetic and environmental factors. Epigenetic alterations, such as DNA methylation are considered pivotal in the inactivation of tumor-related genes. We assessed a methylation panel of 5 genes to study their association to GC progression and microsatellite instability (MSI), and studied the role of RUNX3 in GC pathogenesis and the tumor immune microenvironment. The methylation status of 47 promoter-CpG islands was studied through MALDI-TOF mass spectrometry analysis in 35 Microsatellite stable (MSS) GC, 26 MSI, and 18 cancer-free samples (CFS), and 6 MSS GC and 4 MSI GC cell lines. We also studied RUNX3 expression by immunohistochemistry (IHC) in 40 samples, and validated differences in methylation levels between tumor, normal, and immune tissue in 14 additional samples. Unsupervised hierarchical clustering of methylation levels revealed no distinct subgroups between MSI and MSS samples or cell lines. CFSs clustered together showing higher levels of RUNX3 methylation compared to GC samples. RUNX3 showed protein silencing in cancer and normal mucosa, compared to inflammatory peritumoural infiltrate in almost all cases, showing a non-lymphocytic predominant pattern and being correlated with epigenetic silencing. Our results show aberrant promoters methylation in APC, CDH1, CDKN2A, MLH1 and RUNX3 associated with GC, as well as a non-lymphocytic predominant infiltrate with high expression of RUNX3. Deep study of RUNX3 inflammation signaling could help in understanding inflammation and immune activation in the tumor microenvironment.

Modeling MC1R rare variants: a structural evaluation of variants detected in a Mediterranean case-control study.

TO THE EDITOR Many diseases are caused by single amino-acid substitutions on key genes because of the loss of stability, protein misfolding, and changes in the interaction properties (Honig and Nicholls, 1995; Goldberg, 2003). Protein structures can be used to evaluate the effect of missense mutations on diseaseassociated proteins, especially on highly polymorphic genes, where functional studies are likely to be incomplete. Considering the involvement of the highly polymorphic melanocortin-1 receptor gene (MC1R) in melanoma predisposition, we investigated the role of the rare variants (minor allele frequency o1%) found in this gene in the Spanish population. A structural analysis was performed by evaluating the amino-acid substitutions in the context of a threedimensional (3D) model and comparing their effects with available functional data. The MC1R gene, which is located on chromosome 16q24.3 and codes for a melanocyte-stimulating hormone receptor with seven-transmembrane helices, is to date the main genetic contributor to sporadic melanoma predisposition (Kennedy et al., 2001). It has already been associated with melanoma in different Caucasian populations (Fernandez et al., 2007; Ibarrola-Villava et al., 2010, 2012). It is highly polymorphic, with more than 100 non-conservative reported variants (Gerstenblith et al., 2007). Most of these changes are relatively rare, but their frequencies vary among populations, and it appears that they are more commonly found in South European populations than in those of North European descent (Gerstenblith et al., 2007; Williams et al., 2011). Of the 1170 individuals sequenced (710 non-related melanoma cases and 460 cancer-free controls), 697 (59.6%) carried at least one MC1R variant, with 78 (11.2%) of them actually carrying a rare variant. Overall, 6.7% (78 of 1170 individuals) of the Spanish population carry a MC1R rare variant. We detected a total of 41 different MC1R variants. Among these, 31 variants appear rarely in Spain and variant Y298H appeared for the first time in our population. We have obtained statistically significant individual associations with melanoma for six variants: V60L, S83P, R151C, I155T, R160W, and D294H (Supplementary Table S1 online). The highest ORs were estimated for I155T and R160W (odds ratio (OR): 3.81; Pvalue1⁄4 0.003 and OR: 3.83; Pvalue1⁄4 0.001, respectively). The estimated OR associated with carrying at least one non-synonymous variant was 1.79 (P-value1⁄4 4.93 10 ). However, the OR for carrying only one rare variant was 0.97 (P-value1⁄40.93). There are many common non-synonymous MC1R variants associated with the red hair color (RHC) phenotype: D84E, R142H, R151C, I155T, R160W, and D294H. The association of other common variants such as V60L, V92M, and R163Q with the RHC phenotype has not been clearly established, and therefore these variants have been considered non-red hair color (NRHC) (Wong and Rees, 2005). Almost all carriers of rare changes in the Spanish population tend to have fair skin color. However, only 40% of them also harbored a RHC or NRHC variant. Thus, rare variants might slightly modulate skin pigmentation. Different studies have demonstrated the functional consequences of some of these MC1R changes. Thus, mutations in positions 84, 142, 151, 160, and 294 have been previously reported to alter protein function (Ringholm et al., 2004; Garcia-Borron et al., 2005; Newton et al., 2007; Sanchez-Laorden et al., 2009; Dessinioti et al., 2011). In addition, previous functional studies have validated some of the 3D model prediction results. Position M128 appears to have no effect according to in silico predictions; however, the 3D model suggests it could be a putative folding disrupter, later confirmed in a functional study (Perez Oliva et al., 2009). Owing to the importance of MC1R variants in pigmentation and melanoma predisposition, we have undertaken a comprehensive 3D structure modeling of 29 non-synonymous changes detected in our population to further explain putative implications of rare variants (Table 1 and Figure 1). After evaluating all changes according to MC1R structure, variant location and nature of the amino-acid changes, 25 variants seemed to have structural implications and cluster in three different regions that may explain their differences in functionality: the GTPase/protein kinase C (PKC) signaling region, an alleged amelanocyte-stimulating hormone (aMSH)-binding extracellular region, and a novel central core protein region. The first and better-known region harbors some of the common RHC (R142H, R151C, I155T, and R160W) and NRHC (R163Q) variants. Although these variants do not seem to bring about structural changes under the 3D model, they are located in an intracellular region thought to be a GTPase and PKC interaction area that initiates the receptor downstream signaling pathway and internalization of the receptor that confirms previously reported functionality Accepted article preview online 11 November 2013; published online 12 December 2013 Abbreviations: 3D, three-dimensional; MC1R, melanocortin-1 receptor gene; MSH, melanocytestimulating hormone; NRHC, non-red hair color; OR, odds ratio; PKC, protein kinase C; RHC, red hair color M Ibarrola-Villava et al. Modeling MC1R Rare Variants

138 Deregulation of ARID1A, CDH1, cMET and PIK3CA and target-related microRNA expression in gastric cancer

Genetic and epigenetic alterations play an important role in gastric cancer (GC) pathogenesis. Aberrations of the phosphatidylinositol-3-kinase signaling pathway are well described. However, emerging genes have been described such as, the chromatin remodeling gene ARID1A. Our aim was to determine the expression levels of four GCrelated genes, ARID1A, CDH1, cMET and PIK3CA, and 14 target-related microRNAs (miRNAs). We compared mRNA and miRNA expression levels among 66 gastric tumor and normal adjacent mucosa samples using quantitative real-time reverse transcription PCR. Moreover, ARID1A, cMET and PIK3CA protein levels were assessed by immunohistochemistry (IHC). Finally, gene and miRNAs associations with clinical characteristics and outcome were also evaluated. An increased cMET and PIK3CA mRNA expression was found in 78.0% (P = 2.20 × 10 −5 ) and 73.8% (P = 1.00 × 10 −3 ) of the tumors, respectively. Moreover, IHC revealed that cMET and PIK3CA expression was positive in 63.6% and 87.8% of the tumors, respectively. Six miRNAs had significantly different expression between paired-samples, finding five up-regulated [miR-223-3p ( P = 1.65 × 10 −6 ), miR-19a3p (P = 1.23 × 10 −4 ), miR-128-3p (P = 3.49 × 10 −4 ), miR-130b-3p (P = 1.00 × 10 −3

Retinal nerve fibre layer and brain grey substance as early prognostic factors for disability in multiple sclerosis

We read with interest the article by Bock et al ,1 in which the association between retinal nerve fibre layer (RNFL) and total macular volume (TMV) was analysed by optical coherence tomography (OCT) and contrast sensitivity was measured by functional acuity contrast testing in relapsing-remitting multiple sclerosis (MS) patients. The authors found that the functional contrast vision in MS is influenced by morphological changes in the anterior visual pathway. This work emphasised that contrast sensitivity is reduced in MS, and that RNFL and TMV, as morphological measures of retinal axon loss, are predictors of contrast sensitivity. Ophthalmological examinations were the basis of the Bock et al 1 study. There …