Guidelines for NGS procedures applied to prenatal diagnosis by the Spanish Society of Gynecology and Obstetrics and the Spanish Association of Prenatal Diagnosis.

OBJECTIVE: This document addresses the clinical application of next-generation sequencing (NGS) technologies for prenatal genetic diagnosis and aims to establish clinical practice recommendations in Spain to ensure uniformity in implementing these technologies into prenatal care. METHODS: A joint committee of expert obstetricians and geneticists was created to review the existing literature on fetal NGS for genetic diagnosis and to make recommendations for Spanish healthcare professionals. RESULTS: This guideline summarises technical aspects of NGS technologies, clinical indications in prenatal setting, considerations regarding findings to be reported, genetic counselling considerations as well as data storage and protection policies. CONCLUSIONS: This document provides updated recommendations for the use of NGS diagnostic tests in prenatal diagnosis. These recommendations should be periodically reviewed as our knowledge of the clinical utility of NGS technologies, applied during pregnancy, may advance.

SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis.

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10-3, 95% confidence interval: 1.5-15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).

Experience using singleton exome sequencing of probands as an approach to preconception carrier screening in consanguineous couples.

BACKGROUND: Consanguineous couples have an increased risk of severe diseases in offspring due to autosomal recessive disorders. Exome sequencing (ES) offers the possibility of extensive preconception carrier screening (PCS) in consanguineous couples who may be at risk of rare genetic disorders. METHODS: We retrospectively analysed ES data from 65 probands affected with rare genetic disorders born from consanguineous couples. We explored diagnostic yield and carrier status for recessive disorders. RESULTS: The overall diagnostic yield in a singleton approach was 53.8%, mostly recessive variants. In a hypothetical exome-based PCS, only 11.7% of these causative rare variants would have been missed in the filtering process. Carrier screening for recessive conditions allowed the identification of at least one additional pathogenic or likely pathogenic variant in 85.7% of the probands, being the majority with a gene carrier frequency <1 in 200. In addition, considering only clinically actionable conditions, we estimated that 12.3% of our close consanguineous couples may be at risk for an additional recessive disease. CONCLUSIONS: Our results demonstrate that ES outperforms panel-based screening in a PCS context in consanguineous couples and could potentially increase their reproductive autonomy and facilitate informed decision-making.

Clinical and psychological implications of secondary and incidental findings in cancer susceptibility genes after exome sequencing in patients with rare disorders.

BACKGROUND/OBJECTIVES: Exome sequencing may identify pathogenic variants unrelated with the purpose of the analysis. We investigated the frequency of secondary and incidental findings (SF/IF) in cancer susceptibility genes (CSG), their clinical actionability and the psychological impact in individuals with an SF/IF (cases) compared with individuals tested due to their cancer history (controls). METHODS: This study analysed 533 exomes ordered for non-cancer conditions. Medical records were reviewed for clinical actionability of SF/IF. Psychological impact was analysed using the Multidimensional Impact of Cancer Risk Assessment (MICRA) scale and compared between cases and controls with a propensity score weighting method. RESULTS: The frequency of SF/IF in CSG was 2.1% (95% CI 1.1% to 3.8%): three BRCA2, three PMS2, two SDHB, and one each in BRCA1, MLH1 and RAD51C. Among the relatives, 18 were carriers. Twenty enrolled for surveillance, and a neoplasm was diagnosed in 20%: three paragangliomas and one breast cancer. Cases presented higher MICRA mean scores than controls (21.3 vs 16.2 in MICRA total score, 6.3 vs 4.2 in the distress subscale, and 8.3 vs 6.6 in the uncertainty subscale; all p<0.001). CONCLUSION: SF/IF in CSG were identified in 2.1% of patients. Despite a numerically higher psychological impact, the identification of SF/IF allowed early detection and cancer prevention in families without cancer history.

An spanish study of secondary findings in families affected with mendelian disorders: choices, prevalence and family history.

Clinical exome sequencing has the potential to identify pathogenic variants unrelated to the purpose of the study (secondary findings, SFs). Data describing actual choices of SFs in participants in a clinical setting and factors influencing their decision are virtually non-existant in Europe. In this work, we report the acceptance rate of SFs, calculate their prevalence and study factors associated with the decision in a cohort of patients affected with a rare genetic disorder in a Spanish Hospital. Finally, we re-examine the presence of previously non reported family history in positive cases. We retrospectively reviewed informed consent choices and SF results from 824 unrelated probands affected with rare genetic disorders who underwent whole-genome or exome sequencing. Ninety percent of families (740/824) affected with rare disorders wished to be informed of SFs. Declining SFs was associated with a prenatal setting (30% vs. 8.7%, p = 0.025), consanguinity (19% vs. 8.7%, p = 0.013), male gender (10.6% vs. 1.5%, p = 0.00865) and the proband being a minor (10.6% vs. 1.5%, p = 0.014). Overall, 27 pathogenic or likely pathogenic variants were identified in 27 individuals, with an SF prevalence of 3.6%. Disclosure of SFs increased the percentage of positive family histories and resulted in early diagnosis or changes in the management of 10 individuals from five families. We show that the acceptance of SFs in Spain is high and the disclosure of SFs leads to a clinically meaningful change in the medical management of individuals.

A method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome.

Methods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Here we describe a method to target, multiplex and sequence at high coverage full-length human mitochondrial genomes as native single-molecules, utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks, that define the mtDNA beginning and end of the sequencing reads, as barcodes, we achieve high demultiplexing specificity and delineation of the full-length of the mtDNA, regardless of the structural variant pattern. The long-read sequencing data is analysed with a pipeline where our custom-developed software, baldur, efficiently detects single nucleotide heteroplasmy to below 1%, physically determines phase and can accurately disentangle complex deletions. Our workflow is a tool for studying mtDNA variation and will accelerate mitochondrial research.

Current Status of Genetic Counselling for Rare Diseases in Spain.

Genetic Counselling is essential for providing personalised information and support to patients with Rare Diseases (RD). Unlike most other developed countries, Spain does not recognize geneticists or genetic counsellors as healthcare professionals Thus, patients with RD face not only challenges associated with their own disease but also deal with lack of knowledge, uncertainty, and other psychosocial issues arising as a consequence of diagnostic delay. In this review, we highlight the importance of genetic counsellors in the field of RD as well as evaluate the current situation in which rare disease patients receive genetic services in Spain. We describe the main units and strategies at the national level assisting patients with RD and we conclude with a series of future perspectives and unmet needs that Spain should overcome to improve the management of patients with RD.

Cap a la genètica assistencial interdisciplinària i transversal: evolució de cinquanta anys en un hospital terciari a partir de la pediatria / Hacia la genètica asistencial interdisciplinaria y transversal: evolución de cincuenta años en un hospital terciario a partir de la pediatría / Towards interdisciplinary and transversal clinical genetics: a 50-year evolution from pediatrics in a tertiary hospital

L’objectiu d’aquest treball és analitzar l’evolució de la demanda assistencial i les possibilitats diagnòstiques, en laconsulta de genètica clínica d’un hospital de tercer nivell durant els últims cinquanta anys i també a partir dels seus inicis com a consulta específica de Pediatria.S’hi analitzen tant els motius de consulta com les proves de laboratori disponibles per arribar al diagnòstic dels pacients valorats durant el període 1968-2018, a la consulta de Genètica Clínica i al Laboratori de Genètica. A partir de 200 consultes anuals, en els primers anys, arribem a l’actualitat, en què se’n fan al voltant de 8.000 (entre primeres, successives i interconsultes), distribuïdes en Genètica Clínica i Assessorament Genètic, fins a un total de més de 32.000 pacients visitats durant aquests cinquanta anys.Al Laboratori de Genètica, l’evolució abasta des de l’estudi del cariotip convencional fins a l’aplicació de les tecnologies genòmiques actuals, i s’hi fan més de 9.000 estudis anuals de pacients de l’Hospital. Amb la incorporació de noves tecnologies moleculars s’ha canviat el paradigma de l’estudi genètic i s’ha aconseguit un rendiment millor: s’han pogut incrementar els diagnòstics i també s’ha reduït el temps necessari per obtenir-los.A més de la transformació del genetista que col·labora en el seguiment multidisciplinari dels pacients, s’evidencia uncanvi i una diversificació del motiu de consulta i s’estableix el valor de la incorporació, a partir del 2010, de professionals especialitzats en assessorament genètic per donar resposta a aquesta demanda.Els canvis experimentats en els motius de consulta, els diagnòstics i les proves de laboratori fetes durant tots aquests anys reflecteixen la importància de la incorporació i la interacció, en una mateixa àrea o unitat assistencial, de professionals especialitzats en genètica clínica, assessorament genètic i laboratori de genètica integral (també ambbioinformàtics). (AU)

El objetivo de este trabajo es analizar la evolución de la demandaasistencial y las posibilidades diagnósticas en la consulta de genética clínica de un hospital de tercer nivel a lo largo de los últimos50 años a partir de sus inicios como una consulta específica dePediatría.Se analizan los motivos de consulta y las pruebas de laboratorio disponibles para llegar al diagnóstico de los pacientes valorados en el período 1968-2018 en la Consulta de Genética Clínica y el Laboratorio de Genética. A partir de 200 consultas anuales en los primeros años llegamos a la actualidad, en que se realizan alrededor de 8.000 visitas (primeras, sucesivas e interconsultas) distribuidas en Genética Clínica y Asesoramiento Genético (32.000pacientes visitados hasta la fecha).En el laboratorio de Genética la evolución abarca desde el estudio del cariotipo convencional hasta la aplicación de las tecnologías genómicas actuales realizando más de 9.000 estudios anuales de pacientes del Hospital. Además de la transformación del genetista clínico colaborando en el seguimiento multidisciplinar de los pacientes, se evidencia un cambio y diversificación del motivo de consulta y se establece el valor de la incorporación de profesionales especializados en asesoramiento genético (a partir de 2010) para dar respuesta a esta demanda. Con la incorporación de nuevas tecnologías moleculares se ha cambiado el paradigma del estudio genético con un incremento importante del rendimiento y mejoría en el tiempo en obtener resultados diagnósticos. Los cambios experimentados en los motivos de consulta, los diagnósticos y las pruebas de laboratorio realizadas a lo largo de estos años reflejan la importancia de la incorporación e interacción, en una misma área/unidad asistencial, de profesionales especializados en genética clínica, asesores genéticos y laboratorio de genética integral (incluyendo bioinformáticos). (AU)

The objective of this work is to analyze the evolution of the demand and the diagnostic capabilities in the clinical genetics service of a tertiary hospital over the last 50 years from its initiationas a specific pediatric consultation. The reasons for consultationare analyzed as well as the laboratory tests available to reach thediagnosis of the patients evaluated in the period 1968-2018 at the Clinical Genetics Service and the Genetics Laboratory. From 200 consultations/year in the first years, we have reachedaround 8,000 visits (first, follow-up, and internal consultations) distributed in Clinical Genetics and Genetic Counseling (32,000patients visited to date).The Genetics Laboratory evolved from the study of the conventional karyotype to the application of state of the art genomic technologies, carrying out more than 9,000 annual studies from patients followed–up in the hospital.In addition to the transformation of the role of the clinical geneticist into a member of the multidisciplinary care team, there isevidence of a change and diversification of the reasons for consultation and in the value of incorporating professionals specializedin genetic counseling (starting in 2010) to respond to this demand. With the incorporation of new molecular technologies, theparadigm of the genetic study has changed, with a significant increase in performance and improving time to diagnostic results.The changes experienced in the reasons for consultation, diagnosesand laboratory tests carried out throughout these years reflect theimportance of the incorporation and interaction, in the same healthcare area or unit, of professionals specialized in clinical geneticsand genetic counseling, with a comprehensive genetics laboratory(including bioinformatics). (AU)

Chromosomal microarray analysis in fetuses with central nervous system anomalies: An 8-year long observational study from a tertiary care university hospital.

OBJECTIVES: To evaluate the prevalence of DNA copy number variants (CNVs) detected with array comparative genomic hybridization (CGH) in fetuses with central nervous system (CNS) anomalies. Secondary objectives were to describe the prevalence of CNV in specific CNS abnormalities, in isolated defects or associated with other malformations or fetal growth restriction (FGR). METHODS: Observational cohort study in 238 fetuses with CNS anomalies in which an array-CGH had been performed between January 2009 and December 2017. Pathogenic CNV and variants of unknown significance (VUS) were reported. RESULTS: Pathogenic CNVs were found in 16/238 cases (6.7%), VUS in 18/238 (7.6%), and normal result in 204/238 (85.7%) cases. Pathogenic CNVs were more frequent in posterior fossa anomalies (cerebellar hypoplasia 33%, megacisterna magna 20%), moderate ventriculomegaly (11%) and spina bifida (3.7%). Pathogenic CNVs and VUS were found in 7/182 (3.8%) and 14/182 (7.7%) cases of isolated anomalies, in 9/49 (18.4%) and 4/49 (8.2%) presenting another malformation, and in 0/7 and 0/7 cases with associated FGR (P = .001, P = .741, respectively). CONCLUSION: These results provide strong evidence toward performing array in fetuses with CNS anomalies, particular in cases of posterior fossa anomalies. The prevalence of pathogenic CNVs is higher in association with other malformations.

Beyond the disease itself: A cross-cutting educational initiative for patients and families with rare diseases.

Rare diseases (RDs) as a whole affect a huge number of individuals although each specific condition comprises a low number of individuals. As a consequence, funds allocated to expand research to all conditions are often limited. Several initiatives have emerged to invest more resources for research in RDs, but patients express unmet needs regarding educational initiatives, awareness support, and psychosocial resources. We developed an educational training program in the format of weekly sessions covering basic medical scientific knowledge and psychosocial aspects of RDs. The aim of this initiative was to assess its overall impact regarding knowledge, psychological issues, and participant satisfaction. Items were evaluated through surveys before and after the sessions. Here, we report the experience and impact of two editions of this initiative with a total of 37 participants. Our results show improvements in knowledge and better management of the psychological impact. Moreover, participants were able to exchange experiences and concerns, most of which were shared even though the RDs were different. Overall, the program was evaluated by the participants as a highly beneficial experience and all of them were interested in attending advanced editions.

Utility of two SMN1 variants to improve spinal muscular atrophy carrier diagnosis and genetic counselling.

Spinal muscular atrophy (SMA) is caused by deletions/mutations in SMN1. Most heterozygous SMA carriers have only one SMN1 copy in one of the alleles (1/0 carriers). However, a few carriers lack SMN1 in one of their chromosomes, but present two gene copies in the other. These "2/0 carriers" are undistinguishable from non-carrier individuals (1/1) with currently available methods. Previous association of SMN1 variants c.*3 + 80 T > G and c.*211_*212del with two SMN1 copies in cis in Ashkenazi population prompted us to analyze them in 270 Spanish individuals (SMA carriers, patients and general population). Both variants were much more frequently detected in chromosomes with 2 SMN1 copies in cis in comparison with chromosomes carrying one copy (17.9 vs. 0.7%; p < 0.001). In particular, one-fifth of 2/0 SMA carriers harboured one or both variants compared to none of 99 non-carriers with two SMN1 copies (p < 0.001). The c.*211_*212del variant was also much more frequent in exon 8 of SMN2-SMN1 hybrids than in that of intact SMN1 genes (20 vs. 0.83%, p < 0.001), suggesting its association with chromosomal rearrangements. Although absence of these variants does not exclude that a particular individual is a 2/0 SMA carrier, their presence is valuable to substantially increase residual risk in putative carriers, thus improving genetic counselling.

Genetic Variants Associated with Colorectal Adenoma Susceptibility.

BACKGROUND: Common low-penetrance genetic variants have been consistently associated with colorectal cancer risk. AIM: To determine if these genetic variants are associated also with adenoma susceptibility and may improve selection of patients with increased risk for advanced adenomas and/or multiplicity (≥ 3 adenomas). METHODS: We selected 1,326 patients with increased risk for advanced adenomas and/or multiplicity and 1,252 controls with normal colonoscopy from population-based colorectal cancer screening programs. We conducted a case-control association study analyzing 30 colorectal cancer susceptibility variants in order to investigate the contribution of these variants to the development of subsequent advanced neoplasia and/or multiplicity. RESULTS: We found that 14 of the analyzed genetic variants showed a statistically significant association with advanced adenomas and/or multiplicity: the probability of developing these lesions increased with the number of risk alleles reaching a 2.3-fold risk increment in individuals with ≥ 17 risk alleles. CONCLUSIONS: Nearly half of the genetic variants associated with colorectal cancer risk are also related to advanced adenoma and/or multiplicity predisposition. Assessing the number of risk alleles in individuals within colorectal cancer screening programs may help to identify better a subgroup with increased risk for advanced neoplasia and/or multiplicity in the general population.

NGS-Based Assay for the Identification of Individuals Carrying Recessive Genetic Mutations in Reproductive Medicine.

Next-generation sequencing (NGS) has the capacity of carrier screening in gamete donation (GD) programs. We have developed and validated an NGS carrier-screening test (qCarrier test) that includes 200 genes associated with 368 disorders (277 autosomal recessive and 37 X-linked). Carrier screening is performed on oocyte donation candidates and the male partner of oocyte recipient. Carriers of X-linked conditions are excluded from the GD program, whereas donors are chosen who do not carry mutations for the same gene/disease as the recipients. The validation phase showed a high sensitivity (>99% sensitivity) detecting all single-nucleotide variants, 13 indels, and 25 copy-number variants included in the validation set. A total of 1,301 individuals were analysed with the qCarrier test, including 483 candidate oocyte donors and 635 receptor couples, 105 females receiving sperm donation, and 39 couples seeking pregnancy. We identified 56% of individuals who are carriers for at least one genetic condition and 1.7% of female donors who were excluded from the program due to a carrier state of X-linked conditions. Globally, 3% of a priori assigned donations had a high reproductive risk that could be minimized after testing. Genetic counselling at different stages is essential for helping to facilitate a successful and healthy pregnancy.

Whole-exome sequencing identifies rare pathogenic variants in new predisposition genes for familial colorectal cancer.

PURPOSE: Colorectal cancer is an important cause of mortality in the developed world. Hereditary forms are due to germ-line mutations in APC, MUTYH, and the mismatch repair genes, but many cases present familial aggregation but an unknown inherited cause. The hypothesis of rare high-penetrance mutations in new genes is a likely explanation for the underlying predisposition in some of these familial cases. METHODS: Exome sequencing was performed in 43 patients with colorectal cancer from 29 families with strong disease aggregation without mutations in known hereditary colorectal cancer genes. Data analysis selected only very rare variants (0-0.1%), producing a putative loss of function and located in genes with a role compatible with cancer. Variants in genes previously involved in hereditary colorectal cancer or nearby previous colorectal cancer genome-wide association study hits were also chosen. RESULTS: Twenty-eight final candidate variants were selected and validated by Sanger sequencing. Correct family segregation and somatic studies were used to categorize the most interesting variants in CDKN1B, XRCC4, EPHX1, NFKBIZ, SMARCA4, and BARD1. CONCLUSION: We identified new potential colorectal cancer predisposition variants in genes that have a role in cancer predisposition and are involved in DNA repair and the cell cycle, which supports their putative involvement in germ-line predisposition to this neoplasm.

The transcription factor GATA6 enables self-renewal of colon adenoma stem cells by repressing BMP gene expression.

Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with ß-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.

The MLH1 c.1852_1853delinsGC (p.K618A) variant in colorectal cancer: genetic association study in 18,723 individuals.

Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as "variants of uncertain significance", being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome.

New genes emerging for colorectal cancer predisposition.

Colorectal cancer (CRC) is one of the most frequent neoplasms and an important cause of mortality in the developed world. This cancer is caused by both genetic and environmental factors although 35% of the variation in CRC susceptibility involves inherited genetic differences. Mendelian syndromes account for about 5% of the total burden of CRC, with Lynch syndrome and familial adenomatous polyposis the most common forms. Excluding hereditary forms, there is an important fraction of CRC cases that present familial aggregation for the disease with an unknown germline genetic cause. CRC can be also considered as a complex disease taking into account the common disease-commom variant hypothesis with a polygenic model of inheritance where the genetic components of common complex diseases correspond mostly to variants of low/moderate effect. So far, 30 common, low-penetrance susceptibility variants have been identified for CRC. Recently, new sequencing technologies including exome- and whole-genome sequencing have permitted to add a new approach to facilitate the identification of new genes responsible for human disease predisposition. By using whole-genome sequencing, germline mutations in the POLE and POLD1 genes have been found to be responsible for a new form of CRC genetic predisposition called polymerase proofreading-associated polyposis.

Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements.

Exonic variants can alter pre-mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5' splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers' scores recently established by Ke et al. (). Comparisons of hexamer-based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high-throughput sequencing.

Genetic susceptibility variants associated with colorectal cancer prognosis.

Colorectal cancer (CRC) is the second leading cause of cancer-related death among men and women in Western countries. Once a tumour develops, a differentiated prognosis could be determined by lifestyle habits or inherited and somatic genetic factors. Finding such prognostic factors will be helpful in order to identify cases with a shorter survival or at a higher risk of recurrence that may benefit from more intensive treatment and follow-up surveillance. Sixteen CRC genetic susceptibility variants were directly genotyped in a cohort of 1235 CRC patients recruited by the EPICOLON Spanish consortium. Univariate Cox and multivariate regression analyses were performed taking as primary outcomes overall survival (OS), disease-free survival and recurrence-free interval. Genetic variants rs9929218 at 16q22.1 and rs10795668 at 10p14 may have an effect on OS. The G allele of rs9929218 was linked with a better OS [GG genotype, genotypic model: hazard ratio (HR) = 0.65, 95% confidence interval (CI) 0.45-0.93, P = 0.0179; GG/GA genotypes, dominant model: HR = 0.66, 95% CI 0.47-0.94, P = 0.0202]. Likewise, the G allele of rs10795668 was associated with better clinical outcome (GG genotype, genotypic model: HR = 0.73, 95% CI 0.53-1.01, P = 0.0570; GA genotype, genotypic model: HR = 0.66, 95% CI 0.47-0.92, P = 0.0137; GG/GA genotypes, dominant model: HR = 0.68, 95% CI 0.50-0.94, P = 0.0194). In conclusion, CRC susceptibility variants rs9929218 and rs10795668 may exert some influence in modulating patient's survival and they deserve to be further tested in additional CRC cohorts in order to confirm their potential as prognosis or predictive biomarkers.

A colorectal cancer genome-wide association study in a Spanish cohort identifies two variants associated with colorectal cancer risk at 1p33 and 8p12.

BACKGROUND: Colorectal cancer (CRC) is a disease of complex aetiology, with much of the expected inherited risk being due to several common low risk variants. Genome-Wide Association Studies (GWAS) have identified 20 CRC risk variants. Nevertheless, these have only been able to explain part of the missing heritability. Moreover, these signals have only been inspected in populations of Northern European origin. RESULTS: Thus, we followed the same approach in a Spanish cohort of 881 cases and 667 controls. Sixty-four variants at 24 loci were found to be associated with CRC at p-values <10-5. We therefore evaluated the 24 loci in another Spanish replication cohort (1481 cases and 1850 controls). Two of these SNPs, rs12080929 at 1p33 (Preplication=0.042; Ppooled=5.523x10-03; OR (CI95%)=0.866(0.782-0.959)) and rs11987193 at 8p12 (Preplication=0.039; Ppooled=6.985x10-5; OR (CI95%)=0.786(0.705-0.878)) were replicated in the second Phase, although they did not reach genome-wide statistical significance. CONCLUSIONS: We have performed the first CRC GWAS in a Southern European population and by these means we were able to identify two new susceptibility variants at 1p33 and 8p12 loci. These two SNPs are located near the SLC5A9 and DUSP4 loci, respectively, which could be good functional candidates for the association signals. We therefore believe that these two markers constitute good candidates for CRC susceptibility loci and should be further evaluated in other larger datasets. Moreover, we highlight that were these two SNPs true susceptibility variants, they would constitute a decrease in the CRC missing heritability fraction.