Association Between Germline Mutations in BRF1, a Subunit of the RNA Polymerase III Transcription Complex, and Hereditary Colorectal Cancer.

BACKGROUND & AIMS: Although there is a genetic predisposition to colorectal cancer (CRC), few of the genes that affect risk have been identified. We performed whole-exome sequence analysis of individuals in a high-risk family without mutations in genes previously associated with CRC risk to identify variants associated with inherited CRC. METHODS: We collected blood samples from 3 relatives with CRC in Spain (65, 62, and 40 years old at diagnosis) and performed whole-exome sequence analyses. Rare missense, truncating or splice-site variants shared by the 3 relatives were selected. We used targeted pooled DNA amplification followed by next generation sequencing to screen for mutations in candidate genes in 547 additional hereditary and/or early-onset CRC cases (502 additional families). We carried out protein-dependent yeast growth assays and transfection studies in the HT29 human CRC cell line to test the effects of the identified variants. RESULTS: A total of 42 unique or rare (population minor allele frequency below 1%) nonsynonymous genetic variants in 38 genes were shared by all 3 relatives. We selected the BRF1 gene, which encodes an RNA polymerase III transcription initiation factor subunit for further analysis, based on the predicted effect of the identified variant and previous association of BRF1 with cancer. Previously unreported or rare germline variants in BRF1 were identified in 11 of 503 CRC families, a significantly greater proportion than in the control population (34 of 4300). Seven of the identified variants (1 detected in 2 families) affected BRF1 mRNA splicing, protein stability, or expression and/or function. CONCLUSIONS: In an analysis of families with a history of CRC, we associated germline mutations in BRF1 with predisposition to CRC. We associated deleterious BRF1 variants with 1.4% of familial CRC cases, in individuals without mutations in high-penetrance genes previously associated with CRC. Our findings add additional evidence to the link between defects in genes that regulate ribosome synthesis and risk of CRC.

New insights into POLE and POLD1 germline mutations in familial colorectal cancer and polyposis.

Germline mutations in DNA polymerase ɛ (POLE) and δ (POLD1) have been recently identified in families with multiple colorectal adenomas and colorectal cancer (CRC). All reported cases carried POLE c.1270C>G (p.Leu424Val) or POLD1 c.1433G>A (p.Ser478Asn) mutations. Due to the scarcity of cases reported so far, an accurate clinical phenotype has not been defined. We aimed to assess the prevalence of these recurrent mutations in unexplained familial and early-onset CRC and polyposis, and to add additional information to define the clinical characteristics of mutated cases. A total of 858 familial/early onset CRC and polyposis patients were studied: 581 familial and early-onset CRC cases without mismatch repair (MMR) deficiency, 86 cases with MMR deficiency and 191 polyposis cases. Mutation screening was performed by KASPar genotyping assays and/or Sanger sequencing of the involved exons. POLE p.L424V was identified in a 28-year-old polyposis and CRC patient, as a de novo mutation. None of the 858 cases studied carried POLD1 p.S478N. A new mutation, POLD1 c.1421T>C (p.Leu474Pro), was identified in a mismatch repair proficient Amsterdam II family. Its pathogenicity was supported by cosegregation in the family, in silico predictions, and previously published yeast assays. POLE and POLD1 mutations explain a fraction of familial CRC and polyposis. Sequencing the proofreading domains of POLE and POLD1 should be considered in routine genetic diagnostics. Until additional evidence is gathered, POLE and POLD1 genetic testing should not be restricted to polyposis cases, and the presence of de novo mutations, considered.

Germline Mutations in FAN1 Cause Hereditary Colorectal Cancer by Impairing DNA Repair.

Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.

POLE and POLD1 mutations in 529 kindred with familial colorectal cancer and/or polyposis: review of reported cases and recommendations for genetic testing and surveillance.

PURPOSE: Germ-line mutations in the exonuclease domains of POLE and POLD1 have been recently associated with polyposis and colorectal cancer (CRC) predisposition. Here, we aimed to gain a better understanding of the phenotypic characteristics of this syndrome to establish specific criteria for POLE and POLD1 mutation screening and to help define the clinical management of mutation carriers. METHODS: The exonuclease domains of POLE and POLD1 were studied in 529 kindred, 441 with familial nonpolyposis CRC and 88 with polyposis, by using pooled DNA amplification and massively parallel sequencing. RESULTS: Seven novel or rare genetic variants were identified. In addition to the POLE p.L424V recurrent mutation in a patient with polyposis, CRC and oligodendroglioma, six novel or rare POLD1 variants (four of them, p.D316H, p.D316G, p.R409W, and p.L474P, with strong evidence for pathogenicity) were identified in nonpolyposis CRC families. Phenotypic data from these and previously reported POLE/POLD1 carriers point to an associated phenotype characterized by attenuated or oligo-adenomatous colorectal polyposis, CRC, and probably brain tumors. In addition, POLD1 mutations predispose to endometrial and breast tumors. CONCLUSION: Our results widen the phenotypic spectrum of the POLE/POLD1-associated syndrome and identify novel pathogenic variants. We propose guidelines for genetic testing and surveillance recommendations.Genet Med 18 4, 325-332.

[Generalized secondary peritonitis: predictors of in-hospital mortality and survival and mortality evolutive links]. / Peritonitis secundaria generalizada: predictores de mortalidad y sobrevida, y vinculantes evolutivos de mortalidad.

BACKGROUND: The knowledge of mortality predictors and evolutive variables linked with in-hospital death can help us to optimize corrective procedures. OBJECTIVES: To identify independent predictors of in-hospital mortality and survival, and independent evolutive links with death in patients with generalized secondary peritonitis (GSP). METHODS: Two hundred and forty-two patients admitted into the hospital due to GSP were followed until in-hospital death or hospital discharge. Mortality and survival predictors were identified from several variables evaluated at the time of admission and evolutive links to death from evolutive variables. RESULTS: In-hospital mortality was 16.5%. Independent mortality predictors were APACHE II > or =16 [OR=31,9 (IC 95% 10.5-96,5)] and chronic renal failure history, with specificity (1) An appropriate nutritional condition was the only survival predictor [OR = 0.2 (IC 95% 0.1-0.6)]. The highest contribution to APACHE II predictive power came from the deterioration of blood tests values and vital signs, followed by age, sensory condition, and medical history. Independent evolutive links to mortality were multiorganic dysfunction involving three or more organs [OR = 63.2 (IC 95% 18.4-217)], hemodynamic failure and septic shock, and necessity of vital support with mechanical ventilation and/or inotropic/vasoconstrictor drugs. CONCLUSIONS: In generalized secondary peritonitis the independent predictors of in-hospital mortality are APACHE II score > or =16 and chronic renal failure history, not very useful due to the low prevalence. The only independent survival predictor is an appropriate nutritional status. The evolutive links to mortality are multiorganic dysfunction involving three or more organs, hemodynamic failure and septic shock, and necessity of vital-support with mechanical ventilation and/or inotropic/vasoconstrictor agents.

[Predictors of intrahospitalary mortality in the upper gastrointestinal variceal bleeding due to chronic liver disease treated endoscopically]. / Predictores de mortalidad hospitalaria en la hemorragia digestiva alta variceal por hepatopatía crónica tratada endoscópicamente.

UNLABELLED: Upper gastrointestinal variceal bleeding is one of the most serious complications in patients with chronic liver disease. The aim of this trial is to identify in hospital mortality predictors in this illness. MATERIAL AND METHODS: 106 hospitalizations due to this disease from October 2001 to April 2006 in cohort design. In hospital mortality was confronted with age, sex, liver disease etiology, Apache II score at admission, variceal severity, severity of the hepatic failure evaluated according to the Child Pugh's classes, a history of variceal bleeding, initial endoscopic treatment failure, haemostatic failure within the first 48 hours, and rebleeding after 48 hours from admission. In order to identify independent mortality predictors, all the variables correlated significantly with hospital mortality were selected. For the quantitative variable APACHE II score at admission, the best mortality discrimination value was chosen. RESULTS: independent in hospital mortality predictors were: initial endoscopic treatment failure (p = 0.005), haemostatic failure in the first 48 hours (p = 0.012), and Child Pugh C class (p = 0.024). Although male sex, Apache II score at admission and rebleeding after 48 hours were also significantly related to mortality by univaried model, they did not qualify as independent predictors. CONCLUSIONS: the independent predictors of intrahospitalary mortality in patients with variceal bleeding due to chronic liver disease, and first-line of endoscopic treatment were: 1) Initial haemostatic endoscopic treatment failure, 2) Haemostatic failure in the first 48 hs, and 3) Child Pugh C class hepatic failure at admission.

Exome Sequencing Reveals AMER1 as a Frequently Mutated Gene in Colorectal Cancer.

PURPOSE: Somatic mutations occur at early stages of adenoma and accumulate throughout colorectal cancer progression. The aim of this study was to characterize the mutational landscape of stage II tumors and to search for novel recurrent mutations likely implicated in colorectal cancer tumorigenesis. EXPERIMENTAL DESIGN: The exomic DNA of 42 stage II, microsatellite-stable colon tumors and their paired mucosae were sequenced. Other molecular data available in the discovery dataset [gene expression, methylation, and copy number variations (CNV)] were used to further characterize these tumors. Additional datasets comprising 553 colorectal cancer samples were used to validate the discovered mutations. RESULTS: As a result, 4,886 somatic single-nucleotide variants (SNV) were found. Almost all SNVs were private changes, with few mutations shared by more than one tumor, thus revealing tumor-specific mutational landscapes. Nevertheless, these diverse mutations converged into common cellular pathways, such as cell cycle or apoptosis. Among this mutational heterogeneity, variants resulting in early stop codons in the AMER1 (also known as FAM123B or WTX) gene emerged as recurrent mutations in colorectal cancer. Losses of AMER1 by other mechanisms apart from mutations such as methylation and copy number aberrations were also found. Tumors lacking this tumor suppressor gene exhibited a mesenchymal phenotype characterized by inhibition of the canonical Wnt pathway. CONCLUSIONS: In silico and experimental validation in independent datasets confirmed the existence of functional mutations in AMER1 in approximately 10% of analyzed colorectal cancer tumors. Moreover, these tumors exhibited a characteristic phenotype.

Longer telomeres are associated with cancer risk in MMR-proficient hereditary non-polyposis colorectal cancer.

Aberrant telomere length measured in blood has been associated with increased risk of several cancer types. In the field of hereditary non-polyposis colorectal cancer (CRC), and more particularly in Lynch syndrome, caused by germline mutations in the mismatch repair (MMR) genes, we recently found that cancer-affected MMR gene mutation carriers had shorter telomeres and more pronounced shortening of telomere length with age than controls and unaffected MMR gene mutation carriers. Here we evaluate blood telomere length in MMR-proficient hereditary non-polyposis CRC, i.e. familial CRC type X (fCRC-X). A total of 57 cancer-affected and 57 cancer-free individuals from 34 Amsterdam-positive fCRC-X families were analyzed and compared to the data previously published on 144 cancer-affected and 100 cancer-free MMR gene mutation carriers, and 234 controls. Relative telomere length was measured using a monochrome multiplex quantitative PCR method, following strict measures to avoid sources of bias and adjusting by age. Despite the retrospective nature of our study, the results show that longer telomeres associate with cancer risk in fCRC-X, thus identifying different patterns of telomere length according to the status of the MMR system.

Comprehensive molecular characterisation of hereditary non-polyposis colorectal tumours with mismatch repair proficiency.

BACKGROUND: Hereditary non-polyposis colorectal cancer (CRC) without mismatch repair (MMR) defects occurs in almost half of high-risk CRC families, but its genetic cause(s) is(are) still unknown. We aimed to identify unique molecular features that differentiate hereditary from sporadic MMR-proficient colorectal tumours. METHODS: Genomic alterations in 16 tumours from 14 Amsterdam I-II families were studied using the genome-wide copy number OncoScan™ FFPE microarray. Somatic mutation hotspots in BRAF, KRAS, PIK3CA and TP53 were analysed in 37 colorectal tumours from 26 families and in 99 sporadic MMR-proficient CRCs, using direct automated sequencing and KASPar genotyping assays. CpG methylation index was studied in 25 tumours from 19 families by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). RESULTS: Our findings indicate that hereditary MMR-proficient tumours have overlapping genomic profiles to those obtained in sporadic cases, both suggestive of high chromosomal instability, and no high CpG methylation index. Nevertheless, we identified a significant increase in the frequency of chromosome 2p and 2q gains, and of 10 q loss in Amsterdam I families, as well as low frequency of >2 Mb copy-neutral or -gained loss of heterozygosity (LOH). No statistically significant differences in the frequency of BRAF, KRAS, PIK3CA and TP53 mutations or in the gene mutation patterns were observed. However, TP53 mutations appeared almost twice more frequently in sporadic tumours. CONCLUSIONS: Overall, hereditary MMR-proficient CRCs display similar molecular characteristics than their sporadic counterparts. However, the differences identified, such as the chromosome 2 gain, 10 q loss, or the under-representation of TP53 mutations, if validated in larger series, might be of relevance in the clinical setting and/or in the identification of germline defects underlying some of these familial cases.

Telomere length and genetic anticipation in Lynch syndrome.

Telomere length variation has been associated with increased risk of several types of tumors, and telomere shortening, with genetic anticipation in a number of genetic diseases including hereditary cancer syndromes. No conclusive studies have been performed for Lynch syndrome, a hereditary colorectal cancer syndrome caused by germline mutations in the DNA mismatch repair genes. Here we evaluate telomere length in Lynch syndrome, both as a cancer risk factor and as a mechanism associated with anticipation in the age of cancer onset observed in successive generations of Lynch syndrome families. Leukocyte telomere length was measured in 244 mismatch repair gene mutation carriers from 96 Lynch syndrome families and in 234 controls using a monochrome multiplex quantitative PCR method. Cancer-affected mutation carriers showed significantly shorter telomeres than cancer-free mutation carriers. In addition, cancer-affected carriers showed the most pronounced shortening of telomere length with age, compared with unaffected carriers. The anticipation in the age of cancer onset observed in successive generations was not associated with telomere shortening, although, interestingly, all mother-son pairs showed telomere shortening. In conclusion, cancer-affected mismatch repair gene mutation carriers have distinct telomere-length pattern and dynamics. However, anticipation in the age of onset is not explained by telomere shortening. Pending further study, our findings suggest that telomere attrition might explain the previously reported dependence of cancer risk on the parent-of-origin of mismatch repair gene mutations.

Genetic variant in the telomerase gene modifies cancer risk in Lynch syndrome.

Lynch syndrome (LS) is an inherited cancer-predisposing disorder caused by germline mutations in the mismatch repair (MMR) genes. The high variability in individual cancer risk observed among LS patients suggests the existence of modifying factors. Identifying genetic modifiers of risk could help implement personalized surveillance programs based on predicted cancer risks. Here we evaluate the role of the telomerase (hTERT) rs2075786 SNP as a cancer-risk modifier in LS, studying 255 and 675 MMR gene mutation carriers from Spain and the Netherlands, respectively. The study of the Spanish sample revealed that the minor allele (A) confers increased cancer risk at an early age. The analysis of the Dutch sample confirmed the association of the A allele, especially in homozygosity, with increased cancer risk in mutation carriers under the age of 45 (relative riskLSca<45_AA=2.90; 95% confidence interval=1.02-8.26). Rs2075786 is associated with colorectal cancer (CRC) risk neither in the general population nor in non-Lynch CRC families. In silico studies predicted that the SNP causes the disruption of a transcription binding site for a retinoid receptor, retinoid X receptor alpha, probably causing early telomerase activation and therefore accelerated carcinogenesis. Notably, cancer-affected LS patients with the AA genotype have shorter telomeres than those with GG. In conclusion, MMR gene mutation carriers with hTERT rs2075786 are at high risk to develop a LS-related tumor at an early age. Cancer-preventive measures and stricter cancer surveillance at early ages might help prevent or early detect cancer in these mutation carriers.

Predictores de mortalidad hospitalaria en la hemorragia digestiva alta variceal por hepatopatía crónica tratada endoscópicamente / Predictors of intrahospitalary mortality in the upper gastrointestinal variceal bleeding due to chronic liver disease treated endoscopically

La hemorragia digestiva alta variceal es una de las complicaciones más graves en la hepatopatía crónica. El objetivo de este trabajo es identificar variables que predicen mortalidad hospitalaria. Material y métodos: 106 internaciones por esta patología desde octubre de 2001 hasta abril de 2006 en diseño de cohortes. Se confrontó mortalidad hospitalaria con edad, sexo, etiología de la hepatopatía, score APACHE II al ingreso, imensión de las várices evaluada endoscópicamente, severidad de la insuficiencia hepática evaluada por clases de Child Pugh, antecedente de hemorragia variceal, fracaso de la terapia endoscópica inicial, fracaso hemostático en las primeras 48hs y resangrado posterior a 48hs. Para identificar predictores independientes de mortalidad se seleccionaron todas las variables que correlacionaron significativamente con muerte hospitalaria. Para la variable cuantitativa score APACHE II de ingreso se consideró el valor que mejor discriminó mortalidad. Resultados: los predictores independientes de muerte hospitalaria fueron: el fracaso de la terapia endoscópica inicial (p=0,005), el fracaso hemostático en las primeras 48hs (p=0,012) y la clase C de Child Pugh (p=0,024). Si bien en el sexo masculino el score APACHE II al ingreso y el resangrado con posterioridad a las 48hs también correlacionaron con mortalimortalidad, no calificaron como predictores independientes. Conclusión: en portadores de hepatopatía crónica con hemorragia digestiva alta variceal y primera línea de tratamiento endoscópico, resultaron predictores independientes de muerte hospitalaria: 1) el fracaso de la terapia endoscópica hemostática inicial, 2) el fracaso hemostático en las primeras 48hs, y 3) la clase C de Child Pugh al ingreso.

Upper gastrointestinal variceal bleeding is one of the most serious complications in patients with chronic liver disease. The aim of this trial is to identify in hospital mortality predictors in this illness. Material and methods: 106 hospitalizations due to this disease from October 2001 to April 2006 in cohort design. In hospital mortality was confronted with age, sex, liver disease etiology, Apache II score at admission, variceal severity, severity of the hepatic failure evaluated according to the Child Pugh’s classes, a history of variceal bleeding, initial endoscopic treatment failure, haemostatic failure within the first 48 hours, and rebleeding after 48 hours from admission. In order to identify independent mortality predictors, all the variables correlated significantly with hospital mortality were selected. For the quantitative variable APACHE II score at admission, the best mortality discrimination value was chosen. Results: independent in hospital mortality predictors were: initial endoscopic treatment failure (p = 0,005), haemostatic failure in the first 48 hours (p = 0,012), and Child Pugh C class (p = 0,024). Although male sex, Apache II score at admission and rebleeding after 48 hours were also significantly related to mortality by univaried model, they did not qualify as independent predictors. Conclusions: the independent predictors of intrahospitalary mortality in patients with variceal bleeding due to chronic liver disease, and firstline of endoscopic treatment were: 1) Initial haemostatic endoscopic treatment failure, 2) Haemostatic failure in the first 48hs, and 3) Child Pugh C class hepatic failure at admission.