Gloria Ribas

A common coding variant in CASP8 is associated with breast cancer risk

The Breast Cancer Association Consortium (BCAC) has been established to conduct combined case-control analyses with augmented statistical power to try to confirm putative genetic associations with breast cancer. We genotyped nine SNPs for which there was some prior evidence of an association with breast cancer: CASP8 D302H (rs1045485), IGFBP3 −202 C → A (rs2854744), SOD2 V16A (rs1799725), TGFB1 L10P (rs1982073), ATM S49C (rs1800054), ADH1B 3′ UTR A → G (rs1042026), CDKN1A S31R (rs1801270), ICAM5 V301I (rs1056538) and NUMA1 A794G (rs3750913). We included data from 9–15 studies, comprising 11,391–18,290 cases and 14,753–22,670 controls. We found evidence of an association with breast cancer for CASP8 D302H (with odds ratios (OR) of 0.89 (95% confidence interval (c.i.): 0.85–0.94) and 0.74 (95% c.i.: 0.62–0.87) for heterozygotes and rare homozygotes, respectively, compared with common homozygotes; Ptrend = 1.1 × 10−7) and weaker evidence for TGFB1 L10P (OR = 1.07 (95% c.i.: 1.02–1.13) and 1.16 (95% c.i.: 1.08–1.25), respectively; Ptrend = 2.8 × 10−5). These results demonstrate that common breast cancer susceptibility alleles with small effects on risk can be identified, given sufficiently powerful studies.NOTE: In the version of this article initially published, there was an error that affected the calculations of the odds ratios, confidence intervals, between-study heterogeneity, trend test and test for association for SNP ICAM5 V301I in Table 1 (ICAM5 V301I); genotype counts in Supplementary Table 2 (ICAM5; ICR_FBCS and Kuopio studies) and minor allele frequencies, trend test and odds ratios for heterozygotes and rare homozygotes in Supplementary Table 3 (ICAM5; ICR_FBCS and Kuopio studies). The errors in Table 1 have been corrected in the PDF version of the article. The errors in supplementary information have been corrected online.

Mutation in myosin heavy chain 6 causes atrial septal defect.

Atrial septal defect is one of the most common forms of congenital heart malformation. We identified a new locus linked with atrial septal defect on chromosome 14q12 in a large family with dominantly inherited atrial septal defect. The underlying mutation is a missense substitution, I820N, in α-myosin heavy chain (MYH6), a structural protein expressed at high levels in the developing atria, which affects the binding of the heavy chain to its regulatory light chain. The cardiac transcription factor TBX5 strongly regulates expression of MYH6, but mutant forms of TBX5, which cause Holt-Oram syndrome, do not. Morpholino knock-down of expression of the chick MYH6 homolog eliminates the formation of the atrial septum without overtly affecting atrial chamber formation. These data provide evidence for a link between a transcription factor, a structural protein and congenital heart disease.

SLC45A2: a novel malignant melanoma‐associated gene

Human pigmentation appears to be one of the strongest risk factors for malignant melanoma (MM). In humans, there is a long list of genes known to be involved in rare pigmentary disorders such as albinism. These genes explain most of the variation in pigmentation phenotypes seen in human populations, and they do this by regulating the level of synthesis, chemical composition, packaging, and distribution of melanin. This Spanish case‐control study included 131 consecutive melanoma patients and 245 control subjects frequency‐matched for sex and age. A total of 23 SNPs in six candidate genes (ASP, OCA2, TYR, TYRP1, SILV, and SLC45A) belonging to the pigmentation pathway were genotyped. We found that the variant allele of c.1122C>G, p.Phe374Leu (NCBI dbSNP rs16891982) in SLC45A2 (membrane associated transporter previously known as MATP) was associated with protection from MM (OR, 0.41; 95% CI, 0.24–0.70; P=0.008 after adjustment for multiple testing). This association was validated by the consistent link observed with dark hair, dark skin, dark eye color, and the presence of solar lentigins and childhood sunburns. This is the first time SLC45A2 has been described as a melanoma susceptibility gene in a light‐skinned population. Hum Mutat 0,1–7, 2008.

ERCC4 Associated with Breast Cancer Risk: A Two-Stage Case-Control Study Using High-throughput Genotyping

The failure of linkage studies to identify further high-penetrance susceptibility genes for breast cancer points to a polygenic model, with more common variants having modest effects on risk, as the most likely candidate. We have carried out a two-stage case-control study in two European populations to identify low-penetrance genes for breast cancer using high-throughput genotyping. Single-nucleotide polymorphisms (SNPs) were selected across preselected cancer-related genes, choosing tagSNPs and functional variants where possible. In stage 1, genotype frequencies for 640 SNPs in 111 genes were compared between 864 breast cancer cases and 845 controls from the Spanish population. In stage 2, candidate SNPs identified in stage 1 (nominal P < 0.01) were tested in a Finnish series of 884 cases and 1,104 controls. Of the 10 candidate SNPs in seven genes identified in stage 1, one (rs744154) on intron 1 of ERCC4, a gene belonging to the nucleotide excision repair pathway, was associated with recessive protection from breast cancer after adjustment for multiple testing in stage 2 (odds ratio, 0.57; Bonferroni-adjusted P = 0.04). After considering potential functional SNPs in the region of high linkage disequilibrium that extends across the entire gene and upstream into the promoter region, we concluded that rs744154 itself could be causal. Although intronic, it is located on the first intron, in a region that is highly conserved across species, and could therefore be functionally important. This study suggests that common intronic variation in ERCC4 is associated with protection from breast cancer.

Pigmentation-related genes and their implication in malignant melanoma susceptibility

Abstract:  Human pigmentation appears to be one of the main modulators of individual risk of developing malignant melanoma (MM). A large number of genes are known to be involved in rare pigmentary disorders and explain most of the variation in pigmentation phenotypes seen in human populations. This Spanish case–control study included 205 patients with melanoma and 245 control subjects. Thirty‐one single nucleotide polymorphisms (SNPs) in genes that had been mainly associated with congenital pigmentation syndromes (ADTB3A, ATRN, CHS1, EDNRB, HPS, KIT, MGRN1, MITF, MLANA, MYO5A, MYO7A, OA1, OCA2, PAX3 and SOX10) were selected. We found that the variant allele of OCA2 R419Q (rs1800407) was associated with increased risk of MM (OR 1.55, 95% CI 1.04–2.31, P = 0.03). This effect on melanoma risk appeared to be stronger among individuals with solar lentigines, or at least 50 nevi. We also describe, for the first time, an association with the variant S1666C (rs2276288) in the MYO7A gene (OR 1.35; 95% CI 1.04–1.76; P = 0.03). Again, this association appeared to be stronger in several phenotypic groups such as individuals with fair skin and those with childhood sunburns. We also found that several variants in the pigmentation genes considered were associated with intermediate phenotypic characteristics. Our findings highlight the potential importance of pigmentation genes in sporadic MM susceptibility.

Identification and functional analysis of novel variants of the human melanocortin 1 receptor found in melanoma patients.

The melanocortin 1 receptor, a Gs protein‐coupled receptor expressed in epidermal melanocytes, is a major determinant of skin pigmentation and phototype and an important contributor to melanoma risk. MC1R activation stimulates synthesis of black, strongly photoprotective eumelanin pigments. Several MC1R alleles are associated with red hair, fair skin, increased sensitivity to ultraviolet radiation, and increased skin cancer risk. The MC1R gene is highly polymorphic, but only a few naturally occurring alleles have been functionally characterized, which complicates the establishment of accurate correlations between the signaling properties of mutant alleles and defined cutaneous phenotypes. We report the functional characterization of six MC1R alleles found in Spanish melanoma patients. Two variants (c.152T>C, p.Val51Ala and c.865T>C, p.Cys289Arg) have never been described, and the others (c.112G>A, p.Val38Met; c.122C>T, p.Ser41Phe; c.383T>C, p.Met128Thr; and c.842A>G, p.Asn281Ser) have not been analyzed for function. p.Asn281Ser corresponds to a functionally silent polymorphism. The other mutations are associated with varying degrees of loss of function (LOF), from moderate decreases in coupling to the cAMP pathway (p.Val38Met and p.Val51Ala) to nearly complete absence of functional coupling (p.Ser41Phe, p.Met128Thr, and p.Cys289Arg). The LOF p.Met128Thr and p.Cys289Arg mutants are trafficked to the cell surface, but are unable to bind agonists efficiently. Conversely, LOF of p.Val38Met, p.Ser41Phe, and p.Val51Ala is due to reduced cell surface expression as a consequence of retention in the endoplasmic reticulum (ER). Therefore, LOF of MC1R alleles is frequently associated with aberrant forward trafficking and accumulation within the ER or with inability to bind properly the activatory ligand. Hum Mutat 30:1–12, 2009.

Phenotypic Characterization of Nevus and Tumor Patterns in MITF E318K Mutation Carrier Melanoma Patients

A germline polymorphism of the microphthalmia transcription factor (MITF) gene encoding a SUMOylation-deficient E318K-mutated protein has recently been described as a medium-penetrance melanoma gene. In a clinical assessment of nevi from 301 volunteers taken from Queensland, we identified six individuals as MITF E318K mutation carriers. The phenotype for 5 of these individuals showed a commonality of fair skin, body freckling that varied over a wide range, and total nevus count between 46 and 430; in addition, all were multiple primary melanoma patients. The predominant dermoscopic signature pattern of nevi was reticular, and the frequency of globular nevi in carriers varied, which does not suggest that the MITF E318K mutation acts to force the continuous growth of nevi. Excised melanocytic lesions were available for four MITF E318K carrier patients and were compared with a matched range of wild-type (WT) melanocytic lesions. The MITF staining pattern showed a predominant nuclear signal in all sections, with no significant difference in the nuclear/cytoplasmic ratio between mutation-positive or -negative samples. A high incidence of amelanotic melanomas was found within the group, with three of the five melanomas from one patient suggesting a genetic interaction between the MITF E318K allele and an MC1R homozygous red hair color (RHC) variant genotype.

MC1R variants increased the risk of sporadic cutaneous melanoma in darker-pigmented Caucasians: A pooled-analysis from the M-SKIP project

The MC1R gene is a key regulator of skin pigmentation. We aimed to evaluate the association between MC1R variants and the risk of sporadic cutaneous melanoma (CM) within the M‐SKIP project, an international pooled‐analysis on MC1R, skin cancer and phenotypic characteristics. Data included 5,160 cases and 12,119 controls from 17 studies. We calculated a summary odds ratio (SOR) for the association of each of the nine most studied MC1R variants and of variants combined with CM by using random‐effects models. Stratified analysis by phenotypic characteristics were also performed. Melanoma risk increased with presence of any of the main MC1R variants: the SOR for each variant ranged from 1.47 (95%CI: 1.17–1.84) for V60L to 2.74 (1.53–4.89) for D84E. Carriers of any MC1R variant had a 66% higher risk of developing melanoma compared with wild‐type subjects (SOR; 95%CI: 1.66; 1.41–1.96) and the risk attributable to MC1R variants was 28%. When taking into account phenotypic characteristics, we found that MC1R‐associated melanoma risk increased only for darker‐pigmented Caucasians: SOR (95%CI) was 3.14 (2.06–4.80) for subjects with no freckles, no red hair and skin Type III/IV. Our study documents the important role of all the main MC1R variants in sporadic CM and suggests that they have a direct effect on melanoma risk, independently on the phenotypic characteristics of carriers. This is of particular importance for assessing preventive strategies, which may be directed to darker‐pigmented Caucasians with MC1R variants as well as to lightly pigmented, fair‐skinned subjects.

High stability of microRNAs in tissue samples of compromised quality

Degradation of tissue samples limits performing RNA-based molecular studies, but little is known about the potential usefulness of samples of compromised quality for studies focused on miRNAs. In this work we analyze a series of cryopreserved tissue samples (n = 14), frozen samples that underwent a severe thawing process (n = 10), and their paired formalin-fixed paraffin-embedded (FFPE) tissue samples (n = 24) from patients with breast cancer obtained during primary surgical resection and collected in 2011. Quality and integrity analyses of the total and small fraction of RNA were carried out. Recovery of specific RNA molecules (miRNAs hsa-miR-21, hsa-miR-125b, and hsa-miR-191; snoRNA RNU6B; and mRNAs GAPDH and HPRT1) was also analyzed by quantitative RT-PCR. Our results suggest that visualisation of the small RNA electrophoretic profiles obtained using the Agilent 2100 bioanalyzer makes it possible to differentiate between the three groups of samples (optimally frozen, thawed, and FFPE). We demonstrate that specific miRNA molecules can be similarly recovered from different tissue sample sources, which supports their high degree of stability. We conclude that miRNAs are robustly detected irrespective of the quality of the tissue sample. In this regard, a word of caution should be raised before degraded samples are discarded: although prior quality assessment of the biological material to be analyzed is recommended, our work demonstrates that degraded tissue samples are also suitable for miRNA studies.

MC1R, SLC45A2 and TYR genetic variants involved in melanoma susceptibility in southern European populations: results from a meta-analysis.

BACKGROUND AND METHODS Seven genetic biomarkers previously associated with melanoma were analysed in a meta-analysis conducted in three South European populations: five red hair colour (RHC) MC1R alleles, one SLC45A2 variant (p.Phe374Leu) and one thermosensitive TYR variant (p.Arg402Gln). The study included 1639 melanoma patients and 1342 control subjects. RESULTS The estimated odds ratio (OR) associated with carrying at least one MC1R RHC variant was 2.18 (95% confidence interval (CI): 1.86-2.55; p-value=1.02×10(-21)), with an additive effect for carrying two RHC variants (OR: 5.02, 95% CI: 2.88-8.94, p-value=3.91×10(-8)). The SLC45A2 variant, p.Phe374Leu, was significantly and strongly protective for melanoma in the three South European populations studied, with an overall OR value of 0.41 (95% CI: 0.33-0.50; p-value=3.50×10(-17)). The association with melanoma of the TYR variant p.Arg402Gln was also statistically significant (OR: 1.50; 95% CI: 1.11-2.04; p-value=0.0089). Adjustment for all clinical potential confounders showed that melanoma risks attributable to MC1R and SLC45A2 variants strongly persisted (OR: 2.01 95% CI: 1.49-2.72 and OR: 0.50, 95% CI: 0.31-0.80, respectively), while the association of TYR p.Arg402Gln was no longer significant. In addition, stratification of clinical melanoma risk factors showed that the risk of melanoma was strong in those individuals who did not have clinical risk factors. CONCLUSION In conclusion, our results show without ambiguity that in South European populations, MC1R RHC and SCL45A2 p.Phe374Leu variants are strong melanoma risk predictors, notably in those individuals who would not be identified as high risk based on their phenotypes or exposures alone. The use of these biomarkers in clinical practice could be promising and warrants further discussion.