TAPES: A tool for assessment and prioritisation in exome studies.

Next-generation sequencing continues to grow in importance for researchers. Exome sequencing became a widespread tool to further study the genomic basis of Mendelian diseases. In an effort to identify pathogenic variants, reject benign variants and better predict variant effects in downstream analysis, the American College of Medical Genetics (ACMG) published a set of criteria in 2015. While there are multiple publicly available software's available to assign the ACMG criteria, most of them do not take into account multi-sample variant calling formats. Here we present a tool for assessment and prioritisation in exome studies (TAPES, https://github.com/a-xavier/tapes), an open-source tool designed for small-scale exome studies. TAPES can quickly assign ACMG criteria using ANNOVAR or VEP annotated files and implements a model to transform the categorical ACMG criteria into a continuous probability, allowing for a more accurate classification of pathogenicity or benignity of variants. In addition, TAPES can work with cohorts sharing a common phenotype by utilising a simple enrichment analysis, requiring no controls as an input as well as providing powerful filtering and reporting options. Finally, benchmarks showed that TAPES outperforms available tools to detect both pathogenic and benign variants, while also integrating the identification of enriched variants in study cohorts compared to the general population, making it an ideal tool to evaluate a smaller cohort before using bigger scale studies.

CD36 - a plausible modifier of disease phenotype in familial adenomatous polyposis.

BACKGROUND: Familial adenomatous polyposis (FAP) is a well characterised genetic predisposition to early onset colorectal cancer (CRC) that is characterised by polyposis of the colon and rectum. Animal models have consistently suggested the role of modifier genes in determining disease phenotype, yet none have been substantiated in the human population. The mouse homologue of cluster of differentiation 36 (CD36) has been proposed as a modifier of disease in the MIN mouse model of FAP. METHODS: Three single nucleotide polymorphisms (SNPs); rs1049673, rs1761667 and rs1984112 in CD36, have been investigated in 275 FAP patients to determine if they were associated with age of polyposis or risk of developing disease. RESULTS: The results revealed a substantially lower age of polyposis diagnosis for patients belonging to the severe FAP group (harbouring adenomatous polyposis coli (APC) variants in the mutation cluster region (MCR)) and high age for patients in the attenuated familial adenomatous polyposis (AFAP) group for SNPs rs1761667 and rs1984112. CONCLUSIONS: This study provides evidence for patients belonging to the MCR and AFAP groups harbouring specific genotypes for SNPs in CD36 to initiate screening/treatment for FAP at much earlier (MCR) and much later (AFAP) ages than the norm in today's clinical practice. The findings need to be verified in an independent FAP patient cohort.

Use of multigene-panel identifies pathogenic variants in several CRC-predisposing genes in patients previously tested for Lynch Syndrome.

BACKGROUND: Many families with a high burden of colorectal cancer fulfil the clinical criteria for Lynch Syndrome. However, in about half of these families, no germline mutation in the mismatch repair genes known to be associated with this disease can be identified. The aim of this study was to find the genetic cause for the increased colorectal cancer risk in these unsolved cases. MATERIALS AND METHODS: To reach the aim, we designed a gene panel targeting 112 previously known or candidate colorectal cancer susceptibility genes to screen 274 patient samples for mutations. Mutations were validated by Sanger sequencing and, where possible, segregation analysis was performed. RESULTS: We identified 73 interesting variants, of whom 17 were pathogenic and 19 were variants of unknown clinical significance in well-established cancer susceptibility genes. In addition, 37 potentially pathogenic variants in candidate colorectal cancer susceptibility genes were detected. CONCLUSION: In conclusion, we found a promising DNA variant in more than 25 % of the patients, which shows that gene panel testing is a more effective method to identify germline variants in CRC patients compared to a single gene approach.

The genetic basis of colonic adenomatous polyposis syndromes.

Colorectal cancer (CRC) is one of the most common forms of cancer worldwide and familial adenomatous polyposis (FAP) accounts for approximately 1% of all CRCs. Adenomatous polyposis syndromes can be divided into; familial adenomatous polyposis (FAP) - classic FAP and attenuated familial adenomatous polyposis (AFAP), MUTYH-associated polyposis (MAP), NTHL1-associated polyposis (NAP) and polymerase proofreading-associated polyposis (PPAP). The polyposis syndromes genetics and clinical manifestation of disease varies and cases with clinical diagnosis of FAP might molecularly show a different diagnosis. This review examines different aspects of the adenomatous polyposis syndromes genetics and clinical manifestation of disease; in addition the genotype-phenotype and modifier alleles of FAP will be discussed. New technology has made it possible to diagnose some of the APC mutation negative patients into their respective syndromes. There still remain many molecularly undiagnosed adenomatous polyposis patients indicating that there remain causative genes to be discovered and with today's technology these are expected to be identified in the near future. The knowledge about the role of modifier alleles in FAP will contribute to improved pre-symptomatic diagnosis and treatment. New novel mutations will continually be discovered in genes already associated with disease and new genes will be discovered that are associated with adenomatous polyposis. The search for modifier alleles in FAP should be made a priority.

Expanding the genetic basis of copy number variation in familial breast cancer.

INTRODUCTION: Familial breast cancer (fBC) is generally associated with an early age of diagnosis and a higher frequency of disease among family members. Over the past two decades a number of genes have been identified that are unequivocally associated with breast cancer (BC) risk but there remain a significant proportion of families that cannot be accounted for by these genes. Copy number variants (CNVs) are a form of genetic variation yet to be fully explored for their contribution to fBC. CNVs exert their effects by either being associated with whole or partial gene deletions or duplications and by interrupting epigenetic patterning thereby contributing to disease development. CNV analysis can also be used to identify new genes and loci which may be associated with disease risk. METHODS: The Affymetrix Cytogenetic Whole Genome 2.7 M (Cyto2.7 M) arrays were used to detect regions of genomic re-arrangement in a cohort of 129 fBC BRCA1/BRCA2 mutation negative patients with a young age of diagnosis (<50 years) compared to 40 unaffected healthy controls (>55 years of age). RESULTS: CNV analysis revealed the presence of 275 unique rearrangements that were not present in the control population suggestive of their involvement in BC risk. Several CNVs were found that have been previously reported as BC susceptibility genes. This included CNVs in RPA3, NBN (NBS1), MRE11A and CYP19A1 in five unrelated fBC patients suggesting that these genes are involved in BC initiation and/or progression. Of special interest was the identification of WWOX and FHIT rearrangements in three unrelated fBC patients. CONCLUSIONS: This study has identified a number of CNVs that potentially contribute to BC initiation and/or progression. The identification of CNVs that are associated with known tumour suppressor genes is of special interest that warrants further larger studies to understand their precise role in fBC.

Cell cycle-related genes as modifiers of age of onset of colorectal cancer in Lynch syndrome: a large-scale study in non-Hispanic white patients.

Heterogeneity in age of onset of colorectal cancer in individuals with mutations in DNA mismatch repair genes (Lynch syndrome) suggests the influence of other lifestyle and genetic modifiers. We hypothesized that genes regulating the cell cycle influence the observed heterogeneity as cell cycle-related genes respond to DNA damage by arresting the cell cycle to provide time for repair and induce transcription of genes that facilitate repair. We examined the association of 1456 single nucleotide polymorphisms (SNPs) in 128 cell cycle-related genes and 31 DNA repair-related genes in 485 non-Hispanic white participants with Lynch syndrome to determine whether there are SNPs associated with age of onset of colorectal cancer. Genotyping was performed on an Illumina GoldenGate platform, and data were analyzed using Kaplan-Meier survival analysis, Cox regression analysis and classification and regression tree (CART) methods. Ten SNPs were independently significant in a multivariable Cox proportional hazards regression model after correcting for multiple comparisons (P < 5 × 10(-4)). Furthermore, risk modeling using CART analysis defined combinations of genotypes for these SNPs with which subjects could be classified into low-risk, moderate-risk and high-risk groups that had median ages of colorectal cancer onset of 63, 50 and 42 years, respectively. The age-associated risk of colorectal cancer in the high-risk group was more than four times the risk in the low-risk group (hazard ratio = 4.67, 95% CI = 3.16-6.92). The additional genetic markers identified may help in refining risk groups for more tailored screening and follow-up of non-Hispanic white patients with Lynch syndrome.

Combined analysis of three Lynch syndrome cohorts confirms the modifying effects of 8q23.3 and 11q23.1 in MLH1 mutation carriers.

Two colorectal cancer (CRC) susceptibility loci have been found to be significantly associated with an increased risk of CRC in Dutch Lynch syndrome (LS) patients. Recently, in a combined study of Australian and Polish LS patients, only MLH1 mutation carriers were found to be at increased risk of disease. A combined analysis of the three data-sets was performed to better define this association. This cohort-study includes three sample populations combined totaling 1,352 individuals from 424 families with a molecular diagnosis of LS. Seven SNPs, from six different CRC susceptibility loci, were genotyped by both research groups and the data analyzed collectively. We identified associations at two of the six CRC susceptibility loci in MLH1 mutation carriers from the combined LS cohort: 11q23.1 (rs3802842, HR = 2.68, p ≤ 0.0001) increasing risk of CRC, and rs3802842 in a pair-wise combination with 8q23.3 (rs16892766) affecting age of diagnosis of CRC (log-rank test; p ≤ 0.0001). A significant difference in the age of diagnosis of CRC of 28 years was observed in individuals carrying three risk alleles compared to those with 0 risk alleles for the pair-wise SNP combination. A trend (due to significance threshold of p ≤ 0.0010) was observed in MLH1 mutation carriers towards an increased risk of CRC for the pair-wise combination (p = 0.002). This study confirms the role of modifier loci in LS. We consider that LS patients with MLH1 mutations would greatly benefit from additional genotyping of SNPs rs3802842 and rs16892766 for personalized risk assessment and a tailored surveillance program.

The importance of a large sample cohort for studies on modifier genes influencing disease severity in FAP patients.

BACKGROUND: Familial adenomatous polyposis (FAP) is usually characterised by the appearance of hundreds-to-thousands of adenomas throughout the colon and rectum and if left untreated the condition will develop into CRC with close to 100% penetrance. Germline mutations in the APC gene, which plays an integral role in the Wnt-signalling pathway, have been found to be responsible for 70-90% of FAP cases. Several studies suggest that modifier genes may play an important role in the development of CRC and possible modifiers for FAP have been suggested. Interestingly, a study has found that SNPs within ATP5A1 is associated with raised levels of ATP5A1 expression and high expression levels may facilitate CRC development. We aimed to determine if SNPs in ATP5A1 modify the risk of developing CRC/adenomas in FAP patients. METHODS: Genomic DNA from 139 Australian FAP patients with a germline APC mutation underwent genotyping at the Australian Genome Research Facility (AGRF) utilising iPLEX GOLD chemistry with Sequenom MassArray on an Autoflex Spectrometer for 16 SNPs in the ATP5A1 gene. Association between ages of diagnosis/risk of CRC/adenomas was tested with Kaplan-Meier estimator analysis, logistic regression and cox proportional hazard regression. RESULTS: An association between age of diagnosis of CRC and genotypes was observed for SNP rs2578189 (p = 0.0014), with individuals harbouring the variant genotype developing CRC 29 years earlier than individuals harbouring the wildtype genotype. Individuals harbouring the variant genotype of SNP rs2578189 were also at increased risk of CRC (HR = 13.79, 95% CI = 2.36-80.64, p = 0.004). We used an independent Dutch FAP cohort (n = 427) to validate our results; no association between SNP rs2578189 and CRC was observed. CONCLUSION: These results highlight the difficulties in studying a disease that has a high degree of intervention and also emphasize the importance of large sample sizes when searching for modifier genes in patients with an inherited predisposition to disease. To fully determine if there are genetic modifiers of disease in FAP we would encourage people that are interested in collaborating in future studies into the role of modifier genes in disease expression in FAP to join forces.

MTHFR C677T and A1298C polymorphism's effect on risk of colorectal cancer in Lynch syndrome.

Lynch syndrome (LS) is characterised by an increased risk of developing colorectal cancer (CRC) and other extracolonic epithelial cancers. It is caused by pathogenic germline variants in DNA mismatch repair (MMR) genes or the EPCAM gene, leading to a less functional DNA MMR system. Individuals diagnosed with LS (LS individuals) have a 10-80% lifetime risk of developing cancer. However, there is considerable variability in the age of cancer onset, which cannot be attributed to the specific MMR gene or variant alone. It is speculated that multiple genetic and environmental factors contribute to this variability, including two single nucleotide polymorphisms (SNPs) in the methylenetetrahydrofolate reductase (MTHFR) gene: C677T (rs1801133) and A1298C (rs1801131). By decreasing MTHFR activity, these SNPs theoretically reduce the silencing of DNA repair genes and increase the availability of nucleotides for DNA synthesis and repair, thereby protecting against early-onset cancer in LS. We investigated the effect of these SNPs on LS disease expression in 2,723 LS individuals from Australia, Poland, Germany, Norway and Spain. The association between age at cancer onset and SNP genotype (risk of cancer) was estimated using Cox regression adjusted for gender, country and affected MMR gene. For A1298C (rs1801131), both the AC and CC genotypes were significantly associated with a reduced risk of developing CRC compared to the AA genotype, but no association was seen for C677T (rs1801133). However, an aggregated effect of protective alleles was seen when combining the alleles from the two SNPs, especially for LS individuals carrying 1 and 2 alleles. For individuals with germline pathogenic variants in MLH1, the CC genotype of A1298C was estimated to reduce the risk of CRC significantly by 39% (HR = 0.61, 95% CI 0.42, 0.89, p = 0.011), while for individuals with pathogenic germline MSH2 variants, the AC genotype (compared to AA) was estimated to reduce the risk of CRC by 26% (HR = 0.66, 95% CI 0.53, 0.83, p = 0.01). In comparison, no association was observed for C677T (rs1801133). In conclusion, our study suggests that combining the MMR gene information with the MTHFR genotype, including the aggregated effect of protective alleles, could be useful in developing an algorithm that estimates the risk of CRC in LS individuals.

Colorectal cancer susceptibility loci on chromosome 8q23.3 and 11q23.1 as modifiers for disease expression in Lynch syndrome.

OBJECTIVE: Recently, six colorectal cancer (CRC) susceptibility loci have been identified, and two single-nucleotide polymorphisms (SNPs)--rs16892766 (8q23.3) and rs3802842 (11q23.1)--from two of these regions have been found to be significantly associated with an increased CRC risk in patients with Lynch syndrome. The objective of this study was to genotype nine SNPs within these six loci to confirm previous findings and investigate whether they act as modifiers of disease risk in patients with Lynch syndrome. DESIGN: The patient cohort consisted of 684 mutation-positive patients with Lynch syndrome from 298 Australian and Polish families. Nine SNPs were genotyped: rs16892766 (8q23.3), rs7014346 and rs6983267 (8q24.21), rs10795668 (10p14), rs3802842 (11q23.1), rs10318 and rs4779584 (15q13.3), and rs4939827 and rs4464148 (18q21.1). The data were analysed to investigate possible associations between the presence of variant alleles and the risk of developing disease. RESULTS: An association between SNP rs3802842 on chromosome 11q23.1 and rs16892766 on chromosome 8q23.3 and the risk of developing CRC and age of diagnosis was found in MLH1 mutation carriers. Female MLH1 mutation carriers harbouring the homozygous variant genotype for SNP rs3802842 have the highest risk of developing CRC. When the number of risk alleles for the two SNPs combined was analysed, a difference of 24 years was detected between individuals carrying three risk alleles and those carrying no risk alleles. CONCLUSION: The authors were able to replicate the association between the CRC susceptibility loci on chromosomes 8q23.3 and 11q23 and the risk of developing CRC in patients with Lynch syndrome, but the association could only be detected in MLH1 mutation carriers in this study.

P53 in human melanoma fails to regulate target genes associated with apoptosis and the cell cycle and may contribute to proliferation.

BACKGROUND: Metastatic melanoma represents a major clinical problem. Its incidence continues to rise in western countries and there are currently no curative treatments. While mutation of the P53 tumour suppressor gene is a common feature of many types of cancer, mutational inactivation of P53 in melanoma is uncommon; however, its function often appears abnormal. METHODS: In this study whole genome bead arrays were used to examine the transcript expression of P53 target genes in extracts from 82 melanoma metastases and 6 melanoma cell lines, to provide a global assessment of aberrant P53 function. The expression of these genes was also examined in extracts derived from diploid human melanocytes and fibroblasts. RESULTS: The results indicated that P53 target transcripts involved in apoptosis were under-expressed in melanoma metastases and melanoma cell lines, while those involved in the cell cycle were over-expressed in melanoma cell lines. There was little difference in the transcript expression of P53 target genes between cell lines with null/mutant P53 compared to those with wild-type P53, suggesting that altered expression in melanoma was not related to P53 status. Similarly, down-regulation of P53 by short-hairpin RNA (shRNA) had limited effect on P53 target gene expression in melanoma cells, whereas there were a large number of P53 target genes whose mRNA expression was significantly altered by P53 inhibition in melanocytes. Analysis of whole genome gene expression profiles indicated that the ability of P53 to regulate genes involved in the cell cycle was significantly reduced in melanoma cells. Moreover, inhibition of P53 in melanocytes induced changes in gene expression profiles that were characteristic of melanoma cells and resulted in increased proliferation. Conversely, knockdown of P53 in melanoma cells resulted in decreased proliferation. CONCLUSIONS: These results indicate that P53 target genes involved in apoptosis and cell cycle regulation are aberrantly expressed in melanoma and that this aberrant functional activity of P53 may contribute to the proliferation of melanoma.

A genetic variant in telomerase reverse transcriptase (TERT) modifies cancer risk in Lynch syndrome patients harbouring pathogenic MSH2 variants.

Individuals with Lynch syndrome (LS), have an increased risk of developing cancer. Common genetic variants of telomerase reverse transcriptase (TERT) have been associated with a wide range of cancers, including colorectal cancer (CRC) in LS. We combined genotype data from 1881 LS patients, carrying pathogenic variants in MLH1, MSH2 or MSH6, for rs2075786 (G>A, intronic variant), 1207 LS patients for rs2736108 (C>T, upstream variant) and 1201 LS patients for rs7705526 (C>A, intronic variant). The risk of cancer was estimated by heterozygous/homozygous odds ratio (OR) with mixed-effects logistic regression to adjust for gene/gender/country of sample origin considering family identity. The AA genotype of SNP rs2075786 is associated with 85% higher odds at developing cancer compared to GG genotype in MSH2 pathogenic variant carriers (p = 0.0160). Kaplan-Meier analysis also shows an association for rs2075786; the AA allele for MSH2 variant carriers confers risk for earlier diagnosis of LS cancer (log-rank p = 0.0011). We report a polymorphism in TERT to be a possible modifier of disease risk in MSH2 pathogenic variant carriers. The rs2075786 SNP in TERT is associated with a differential risk of developing cancer for MSH2 pathogenic variant carriers. Use of this information has the potential to personalise screening protocols for LS patients.

Haemochromatosis HFE gene polymorphisms as potential modifiers of hereditary nonpolyposis colorectal cancer risk and onset age.

Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by germline mutations in DNA mismatch repair genes; however, variation in disease expression suggests that there are potential modifying factors. Polymorphisms of the HFE gene, which cause the iron overload disorder hereditary haemochromatosis, have been proposed as potential risk factors for the development of colorectal cancer (CRC). To understand the relationship between HNPCC disease phenotype and polymorphisms of the HFE gene, a total of 362 individuals from Australia and Poland with confirmed causative MMR gene mutations were genotyped for the HFE C282Y and H63D polymorphisms. A significantly increased risk of developing CRC was observed for H63D homozygotes when compared with combined wild-type homozygotes and heterozygotes (hazard ratio = 2.93, p = 0.007). Evidence for earlier CRC onset was also observed in H63D homozygotes with a median age of onset 6 years earlier than wild type or heterozygous participants (44 vs. 50 years of age). This effect was significant by all tests used (log-rank test p = 0.026, Wilcoxon p = 0.044, Tarone-Ware p = 0.035). No association was identified for heterozygosity of either polymorphism and limitations on power-prevented investigation of C282Y homozygosity or compound C282Y/H63D heterozygosity. In the Australian sample only, women had a significantly reduced risk of developing CRC when compared with men (hazard ratio = 0.58, p = 0.012) independent of HFE genotype for either single nucleotide polymorphisms. In conclusion, homozygosity for the HFE H63D polymorphism seems to be a genetic modifier of disease expression in HNPCC. Understanding the mechanisms by which HFE interrelates with colorectal malignancies could lead to reduction of disease risk in HNPCC.

Effects of a fall prevention program in elderly: a pragmatic observational study in two orthopedic departments.

PURPOSE: Falls are a common adverse event experienced by elderly in hospitals. This study assessed the effects of a fall prevention program on the rate of fallers, the patient safety culture, and patient-perceived safety. MATERIALS AND METHODS: Two orthopedic departments in different towns in Norway participated in the study. A comprehensive, multifactorial fall prevention program was implemented in one of the departments, the other one was used for control. The changes in the outcomes in the two departments from before to after the intervention were compared. All patients above 64 years of age admitted to the two departments in a 1-year period before and after the intervention were included. All employees at the two departments were invited to participate in surveys measuring the patient safety culture, and a selection of the patients reported patient-perceived safety. The primary outcome was the rate of fallers. Secondary outcomes were the employees' perceived patient safety culture (measured with the Safety Attitudes Questionnaire) and patient-perceived safety (measured with Norwegian Patient Experience Questionnaire). RESULTS: Falls were registered in 114 out of 3,143 patients (3.6%) with 17,006 days in the hospital. Ten patients had two falls, giving a fall rate of 7.3 falls/1,000 days in the hospital. The number of fallers before and after the intervention in the intervention department were 37/734 (5.04%) and 31/735 (4.22%), P=0.46, and in the control department, 25/811 (3.08%) and 21/863 (2.43%), P=0.46. The difference between the changes in the two departments was not statistically significant; 0.17% (95% CI: -2.49 to 2.84; P=0.90). There were also no significant differences in the changes in patient safety culture and patient-perceived safety. CONCLUSION: The fall prevention program revealed no significant effect on the rate of fallers, the patient safety culture, or patient-perceived safety.

Patient participation in the clinical pathway-Nurses' perceptions of adults' involvement in haemodialysis.

AIM: To develop knowledge of nurses' perceptions of participation for patients treated with haemodialysis and their next of kin. DESIGN: A qualitative study with a hermeneutic approach. METHODS: The data were collected in 2015 through focus groups with 13 nurses in Central Norway. RESULTS: The nurses reported that patient participation ranging from non-involvement to shared decision-making was related to whether dialysis was initiated as acute or scheduled. The restrictions required in chronic haemodialysis limited participation. The next of kin were not involved. The nurses highlighted interventions on both the individual and system levels to strengthen participation. CONCLUSION: Dialysis units should develop strategies for participation related to individual needs and design treatment in cooperation with patients and their families, ensuring involvement early in the clinical pathway. Further research is needed on issues related to next of kin, including their desired level of involvement.

Comprehensive mismatch repair gene panel identifies variants in patients with Lynch-like syndrome.

BACKGROUND: Lynch-like syndrome (LLS) represents around 50% of the patients fulfilling the Amsterdam Criteria II/revised Bethesda Guidelines, characterized by a strong family history of Lynch Syndrome (LS) associated cancer, where a causative variant was not identified during genetic testing for LS. METHODS: Using data extracted from a larger gene panel, we have analyzed next-generation sequencing data from 22 mismatch repair (MMR) genes (MSH3, PMS1, MLH3, EXO1, POLD1, POLD3 RFC1, RFC2, RFC3, RFC4, RFC5, PCNA, LIG1, RPA1, RPA2, RPA3, POLD2, POLD4, MLH1, MSH2, MSH6, and PMS2) in 274 LLS patients. Detected variants were annotated and filtered using ANNOVAR and FILTUS software. RESULTS: Thirteen variants were revealed in MLH1, MSH2, and MSH6, all genes previously linked to LS. Five additional genes (EXO1, POLD1, RFC1, RPA1, and MLH3) were found to harbor 11 variants of unknown significance in our sample cohort, two of them being frameshift variants. CONCLUSION: We have shown that other genes associated with the process of DNA MMR have a high probability of being associated with LLS families. These findings indicate that the spectrum of genes that should be tested when considering an entity like Lynch-like syndrome should be expanded so that a more inclusive definition of this entity can be developed.

Lynch syndrome mutation spectrum in New South Wales, Australia, including 55 novel mutations.

BACKGROUND: Lynch syndrome, the most frequent hereditary colorectal cancer syndrome, is caused by defects in mismatch repair genes. Genetic testing is important in order to identify mutation carriers who can benefit from intensive surveillance programs. One of the challenges with genetic testing is the interpretation of pathogenicity of detected DNA variants. The aim of this study was to investigate all putative pathogenic variants tested for at the Division of Molecular Medicine, Pathology North, in Newcastle, Australia, to establish whether previous variant classification is in accordance with that recently performed in the InSiGHT collaboration. METHODS: Prediction programs and available literature were used to classify new variants or variants without classification. RESULTS: We identified 333 mutation positive families, in which 211 different putative pathogenic mismatch repair mutations were found. Most variants with an InSiGHT classification (141 out of 146) were in accordance with our classification. Five variants were discordant, of which one can definitively be reclassified according to the InSiGHT scheme as class 5. Sixty-four variants had not been classified by InSiGHT, of whom 55 have not been previously reported. CONCLUSION: In conclusion, we found that our classifications were mostly in accordance with the InSiGHT scheme. In addition to already known MMR mutations, we have also presented 55 novel pathogenic or putative pathogenic mutations.

Copy number variants associated with 18p11.32, DCC and the promoter 1B region of APC in colorectal polyposis patients.

Familial Adenomatous Polyposis (FAP) is the second most common inherited predisposition to colorectal cancer (CRC) associated with the development of hundreds to thousands of adenomas in the colon and rectum. Mutations in APC are found in ~ 80% polyposis patients with FAP. In the remaining 20% no genetic diagnosis can be provided suggesting other genes or mechanisms that render APC inactive may be responsible. Copy number variants (CNVs) remain to be investigated in FAP and may account for disease in a proportion of polyposis patients. A cohort of 56 polyposis patients and 40 controls were screened for CNVs using the 2.7M microarray (Affymetrix) with data analysed using ChAS (Affymetrix). A total of 142 CNVs were identified unique to the polyposis cohort suggesting their involvement in CRC risk. We specifically identified CNVs in four unrelated polyposis patients among CRC susceptibility genes APC, DCC, MLH1 and CTNNB1 which are likely to have contributed to disease development in these patients. A recurrent deletion was observed at position 18p11.32 in 9% of the patients screened that was of particular interest. Further investigation is necessary to fully understand the role of these variants in CRC risk given the high prevalence among the patients screened.

Targeted next-generation sequencing of 22 mismatch repair genes identifies Lynch syndrome families.

Causative germline mutations in mismatch repair (MMR) genes can only be identified in ~50% of families with a clinical diagnosis of the inherited colorectal cancer (CRC) syndrome hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome (LS). Identification of these patients are critical as they are at substantially increased risk of developing multiple primary tumors, mainly colorectal and endometrial cancer (EC), occurring at a young age. This demonstrates the need to develop new and/or more thorough mutation detection approaches. Next-generation sequencing (NGS) was used to screen 22 genes involved in the DNA MMR pathway in constitutional DNA from 14 HNPCC and 12 sporadic EC patients, plus 2 positive controls. Several softwares were used for analysis and functional annotation. We identified 5 exonic indel variants, 42 exonic nonsynonymous single-nucleotide variants (SNVs) and 1 intronic variant of significance. Three of these variants were class 5 (pathogenic) or class 4 (likely pathogenic), 5 were class 3 (uncertain clinical relevance) and 40 were classified as variants of unknown clinical significance. In conclusion, we have identified two LS families from the sporadic EC patients, one without a family history of cancer, supporting the notion for universal MMR screening of EC patients. In addition, we have detected three novel class 3 variants in EC cases. We have, in addition discovered a polygenic interaction which is the most likely cause of cancer development in a HNPCC patient that could explain previous inconsistent results reported on an intronic EXO1 variant.