The Influence of Smoking and Occupational Risk Factors on DNA Methylation in the AHRR and F2RL3 Genes.

BACKGROUND: AHRR and F2RL3 hypomethylation has been associated with lung cancer. In this study, we investigated the cross-sectional association between smoking and occupational exposures, and AHRR and F2RL3 methylation. METHODS: A case-control study was nested in CARTaGENE to examine the association between AHRR and F2RL3 methylation and lung cancer risk (200 cases; 400 controls). A secondary analysis was conducted using the data collected from this nested study; namely, baseline information on participants' smoking behavior and longest-held job was obtained. A cumulative smoking index summarized information on the number of cigarettes smoked, duration of smoking, and time since cessation. Exposure to 13 occupational agents was estimated using the Canadian Job Exposure Matrix. In baseline blood samples, methylation ratios of 40 CpG sites in the AHRR and F2RL3 genes were measured using Sequenom EpiTYPER. Separate least squares regression models were used to estimate the associations between smoking and occupational exposures, and average AHRR and F2RL3 methylation levels, while adjusting for confounders identified from directed acyclic graphs. RESULTS: In both genes, smoking was associated with lower average methylation levels. Occupational exposure to aromatic amines, cadmium, and formaldehyde were associated with lower AHRR methylation while, only benzene was associated with F2RL3 hypomethylation; these associations were stronger among ever smokers. CONCLUSIONS: Our findings support that smoking and occupational exposures to some agents are associated with AHRR and F2RL3 hypomethylation. IMPACT: Our results inform on mechanisms underlying environmental exposures in lung cancer etiology; future studies should prioritize studying joint exposures.

Data sharing to improve concordance in variant interpretation across laboratories: results from the Canadian Open Genetics Repository.

BACKGROUND: This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation. METHODS: Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin. RESULTS: Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants. CONCLUSIONS: The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.

CCMG practice guideline: laboratory guidelines for next-generation sequencing.

PurposeThe purpose of this document is to provide guidance for the use of next-generation sequencing (NGS, also known as massively parallel sequencing or MPS) in Canadian clinical genetic laboratories for detection of genetic variants in genomic DNA and mitochondrial DNA for inherited disorders, as well as somatic variants in tumour DNA for acquired cancers. They are intended for Canadian clinical laboratories engaged in developing, validating and using NGS methods. METHODS OF STATEMENT DEVELOPMENT: The document was drafted by the Canadian College of Medical Geneticists (CCMG) Ad Hoc Working Group on NGS Guidelines to make recommendations relevant to NGS. The statement was circulated for comment to the CCMG Laboratory Practice and Clinical Practice committees, and to the CCMG membership. Following incorporation of feedback, the document was approved by the CCMG Board of Directors. DISCLAIMER: The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. The current CCMG Practice Guidelines were developed as a resource for clinical laboratories in Canada and should not be considered to be inclusive of all information laboratories should consider in the validation and use of NGS for a clinical laboratory service.

Data sharing as a national quality improvement program: reporting on BRCA1 and BRCA2 variant-interpretation comparisons through the Canadian Open Genetics Repository (COGR).

PurposeThe purpose of this study was to develop a national program for Canadian diagnostic laboratories to compare DNA-variant interpretations and resolve discordant-variant classifications using the BRCA1 and BRCA2 genes as a case study.MethodsBRCA1 and BRCA2 variant data were uploaded and shared through the Canadian Open Genetics Repository (COGR; http://www.opengenetics.ca). A total of 5,554 variant observations were submitted; classification differences were identified and comparison reports were sent to participating laboratories. Each site had the opportunity to reclassify variants. The data were analyzed before and after the comparison report process to track concordant- or discordant-variant classifications by three different models.ResultsVariant-discordance rates varied by classification model: 38.9% of variants were discordant when using a five-tier model, 26.7% with a three-tier model, and 5.0% with a two-tier model. After the comparison report process, the proportion of discordant variants dropped to 30.7% with the five-tier model, to 14.2% with the three-tier model, and to 0.9% using the two-tier model.ConclusionWe present a Canadian interinstitutional quality improvement program for DNA-variant interpretations. Sharing of variant knowledge by clinical diagnostic laboratories will allow clinicians and patients to make more informed decisions and lead to better patient outcomes.

Uptake of Predictive Genetic Testing and Cardiac Evaluation for Children at Risk for an Inherited Arrhythmia or Cardiomyopathy.

Predictive genetic testing in minors should be considered when clinical intervention is available. Children who carry a pathogenic variant for an inherited arrhythmia or cardiomyopathy require regular cardiac screening and may be prescribed medication and/or be told to modify their physical activity. Medical genetics and pediatric cardiology charts were reviewed to identify factors associated with uptake of genetic testing and cardiac evaluation for children at risk for long QT syndrome, hypertrophic cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy. The data collected included genetic diagnosis, clinical symptoms in the carrier parent, number of children under 18 years of age, age of children, family history of sudden cardiac arrest/death, uptake of cardiac evaluation and if evaluated, phenotype for each child. We identified 97 at risk children from 58 families found to carry a pathogenic variant for one of these conditions. Sixty six percent of the families pursued genetic testing and 73% underwent cardiac screening when it was recommended. Declining predictive genetic testing was significantly associated with genetic specialist recommendation (p < 0.001) and having an asymptomatic carrier father (p = 0.006). Cardiac evaluation was significantly associated with uptake of genetic testing (p = 0.007). This study provides a greater understanding of factors associated with uptake of genetic testing and cardiac evaluation in children at risk of an inherited arrhythmia or cardiomyopathy. It also identifies a need to educate families about the importance of cardiac evaluation even in the absence of genetic testing.

Genetic and epigenetic variation in the DNMT3B and MTHFR genes and colorectal adenoma risk.

Polymorphisms in DNMT3B and MTHFR have been implicated in cancer etiology; however, it is increasingly clear that gene-specific DNA methylation also affects gene expression. A cross-sectional study (N = 272) investigated the main and joint effects of polymorphisms and DNA methylation in DNMT3B and MTHFR on colorectal adenoma risk. Polymorphisms examined included DNMT3B c.-6-1045G > T, and MTHFR c.665C > T and c.1286A > C. DNA methylation of 66 and 28 CpG sites in DNMT3B and MTHFR, respectively, was quantified in blood leukocytes using Sequenom EpiTYPER®. DNA methylation was conceptualized using two approaches: (1) average methylation and (2) unsupervised principal component analysis to identify variables that represented methylation around the transcription start site and the gene coding area of both genes. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) associated with the main and joint effects of polymorphisms and DNA methylation. DNA methyltransferase 3B (DNMT3B) TT versus GG/GT genotypes was associated with increased colorectal adenoma risk (OR = 2.12; 95% CI: 1.03-4.34). In addition, increasing DNA methylation in the gene-coding area of DNMT3B was associated with higher risk of colorectal adenomas (OR = 1.34; 95% CI: 1.01-1.79 per SD). In joint effect analyses, synergistic effects were observed among those with both the DNMT3B TT genotype and higher DNMT3B methylation levels compared to those with GT/GG genotypes and lower methylation levels (OR = 4.19; 95% CI: 1.45-12.13 for average methylation; OR = 4.26; 95%CI: 1.31-13.87 for methylation in the transcription start site). This research provides novel evidence that genetic and epigenetic variations contribute to colorectal adenoma risk, precursor to the majority of colorectal cancer (CRC).

Quantification of gene-specific methylation of DNMT3B and MTHFR using sequenom EpiTYPER®.

Among 272 patients undergoing a screening colonoscopy, DNA methylation of DNMT3B and MTHFR, genes encoding enzymes critical to one-carbon metabolism, was quantified in blood leukocytes using Sequenom EpiTYPER®. DNA methylation was quantified in 66 and 28 CpG sites of DNMT3B and MTHFR respectively, and conceptualized using two approaches. First, measures representing average methylation across all CpG sites were created. Second, unsupervised principal component (PC) analysis was used as a pattern derivation and data-reduction approach, to develop two summary variables (PC1 and PC2). These two summary variables represented methylation around the transcription start site (PC1) and in the gene-coding area (PC2) for both DNMT3B and MTHFR. The data contained in this article presents the variation of methylation levels for individual CpG sites within the DNMT3B and MTHFR genes and possible correlations uncovered using PC analysis. The data are related to the research article "Gene-specific DNA methylation of DNMT3B and MTHFR and colorectal adenoma risk" in Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis.

Gene-specific DNA methylation of DNMT3B and MTHFR and colorectal adenoma risk.

DNA methyltransferase 3B (DNMT3B) and methylenetetrahydrofolate reductase (MTHFR) are genes which encode enzymes critical to one-carbon metabolism. Polymorphisms in these genes have been implicated in colorectal cancer etiology; however, epigenetic modifications such as gene-specific DNA methylation also affect gene expression. DNA methylation of DNMT3B and MTHFR was quantified in blood leukocytes using Sequenom EpiTYPER® among 272 participants undergoing a screening colonoscopy. DNA methylation was quantified in 66 and 28CpG sites of DNMT3B and MTHFR respectively, and conceptualized using two approaches. First, measures representing average methylation across all CpG sites were created. Second, unsupervised principal component (PC) analysis was used to identify summary variables representing methylation around the transcription start site and in the gene-coding area for both DNMT3B and MTHFR. Logistic regression was used to compare methylation levels between participants diagnosed with colorectal adenoma(s) versus those with a normal colonoscopy via the estimation of odds ratios (ORs) and 95% confidence intervals (95% CIs) for the risk of colorectal adenomas. No association was observed between average DNA methylation of either DNMT3B or MTHFR and colorectal adenoma risk. For DNMT3B, increasing DNA methylation of CpG sites in the gene-coding area was associated with a higher risk of colorectal adenomas (OR=1.34; 95% CI: 1.01-1.79 per SD). This research provides preliminary evidence that methylation of DNMT3B may have functional significance with respect to colorectal adenomas, precursors to the vast majority of colorectal cancers.

A cross-sectional study of global DNA methylation and risk of colorectal adenoma.

BACKGROUND: The methylation of DNA is recognized as a key epigenetic mechanism and evidence for its role in the development of several malignancies is accumulating. We evaluated the relationship between global methylation in DNA derived from normal appearing colon mucosal tissue and blood leukocytes, and colorectal adenoma risk. METHODS: Patients, aged 40 to 65, scheduled for a screening colonoscopy were recruited. During the colonoscopy, two pinch biopsies of healthy, normal appearing mucosa were obtained from the descending colon. A fasting blood sample was also collected. The methylation status of LINE-1 (long interspersed nuclear element-1) repetitive sequences, as a surrogate measure of global methylation, was quantified in DNA extracted from normal colon mucosa and blood leukocytes. Statistical analysis of the relationship between global DNA methylation and adenoma risk was conducted on 317 participants, 108 subjects with at least one pathologically confirmed adenoma and 209 subjects with a normal colonoscopy. RESULTS: A statistically significant inverse relationship was observed between LINE-1 methylation in colon tissue DNA and adenoma risk for males and for both sexes combined for the lowest methylation quartile compared to the highest (adjusted ORs = 2.94 and 2.26 respectively). For blood, although the overall pattern of odds ratio estimates was towards an increase in risk for lower methylation quartiles compared to the highest methylation quartile, there were no statistically significant relationships observed. A moderate correlation was found between LINE-1 methylation levels measured in tissue and blood (Pearson correlation 0.36). CONCLUSIONS: We observed that lower levels of LINE-1 DNA methylation in normal appearing background colon mucosa were associated with increased adenoma risk for males, and for both sexes combined. Though these findings provide some support for a relationship between LINE-1 DNA methylation in colon mucosal tissue and adenoma risk, large prospective cohort studies are needed to confirm results. Until such investigations are done, the clinical usefulness of LINE-1 methylation as a biomarker of increased adenoma risk is uncertain. Regardless, this study contributes to a better understanding of the role of global DNA methylation as an early event in CR carcinogenesis with implications for future etiologic research.

Discrepant HIV results resolved by human DNA testing.

A high-risk patient was informed of a positive HIV antibody/antigen test. However, follow-up samples taken 2-3 months later for HIV RNA and anti-HIV antibodies were negative. Human DNA testing confirmed that all samples were from this patient, excluding a sample mix-up. Laboratory investigations revealed a likely splash-over contamination event.

Biomarkers measured in buccal and blood leukocyte DNA as proxies for colon tissue global methylation.

There is increasing interest in clarifying the role of global DNA methylation levels in colorectal cancer (CRC) etiology. Most commonly, in epidemiologic studies, methylation is measured in DNA derived from blood leukocytes as a proxy measure of methylation changes in colon tissue. However, little is known about the correlations between global methylation levels in DNA derived from colon tissue and more accessible tissues such as blood or buccal cells. This cross-sectional study utilized DNA samples from a screening colonoscopy population to determine to what extent LINE-1 methylation levels (as a proxy for genome-wide methylation) in non-target tissue (e.g., blood, buccal cells) reflected methylation patterns of colon mucosal tissue directly at risk of developing CRC. The strongest Pearson correlation was observed between LINE-1 methylation levels in buccal and blood leukocyte DNA (r = 0.50; N = 67), with weaker correlations for comparisons between blood and colon tissue (r = 0.36; N = 280), and buccal and colon tissue (r = 0.27; N = 72). These findings of weak/moderate correlations have important implications for interpreting and planning future investigations of epigenetic markers and CRC risk.

A model for GFR alpha 4 function and a potential modifying role in multiple endocrine neoplasia 2.

Mutations of the RET proto-oncogene are found in the majority of patients with the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). A minority of cases, however, have no detectable RET mutation and there is considerable phenotypic variation within and among MEN 2 families with the same RET mutation, suggesting a role for other loci in this disease. A candidate for such a gene is glial cell line-derived neurotrophic factor receptor alpha 4 (GFRA4), which encodes a cell surface-bound co-receptor (GFR alpha 4) required for interaction of RET with its ligand persephin. The GFRA4 gene has multiple alternative splices leading to three distinct protein isoforms that are prominently expressed in thyroid. We postulated that mutations of GFRA4 contribute to MEN 2 in the absence of RET mutations or modify the RET mutation phenotype. We screened patients with MEN 2 or MEN 2-like phenotypes, with and without RET mutations, for variants of GFRA4. We identified 10 variants, one of which was over represented in, and two of which were found exclusively in, our patient populations. One of these was a single-base substitution upstream of the GFR alpha 4 coding region, where it may alter gene expression. The second was a 7 bp insertion, which results in a change in reading frame for all three GFR alpha 4 isoforms. This would cause a relative shift in membrane bound and soluble forms of GFR alpha 4, which would significantly alter the formation of RET signalling complexes. Our data suggest a model of wild-type GFR alpha 4 isoform expression that includes both activating and inhibiting co-receptors for RET.

Paternal transmission of fragile X syndrome.

We present a family in which a fragile X mosaic male, who carries both premutation and full mutation alleles in his peripheral blood leukocytes, has a daughter with both premutation and partially methylated full mutation alleles and a significant developmental disability. To our knowledge, this is the first report of such an occurrence and it challenges current thinking about the expansion and transmission of unstable FMR1 alleles from men to their daughters. It is currently accepted that neither males with premutations nor full mutations are at risk for having daughters with full mutations and fragile X syndrome. The sperm cells of full mutation males are thought to carry only premutation alleles. These alleles, when transmitted through a male, regardless of his cognitive status, are thought to be unable to expand to full mutations in the next generation. In effect, the expansion from premutation to full mutation has only been observed through female meioses. The sperm cells in the father in this family have been shown to contain only alleles in the premutation range. Since his daughter has both premutation and full mutation alleles the expansion to full mutation in this case must have occurred postzygotically.

Boy with 47,XXY,del(15)(q11.2q13) karyotype and Prader-Willi syndrome: a new case and review of the literature.

We report on a 10-year-old boy with a 47,XXY,del(15)(q11.2q13) karyotype and a Prader-Willi syndrome phenotype. His medical history and physical examination conformed to all of the major clinical criteria for Prader-Willi syndrome, but his height was taller than expected based on his hand and foot sizes. The deleted chromosome 15 was paternal in origin and molecular analysis showed maternal origin for the additional X chromosome. These findings suggest that the presence of these two disorders was coincidental in our patient. This supports the findings in the two other 47,XXY and Prader-Willi cases for which parent of origin studies have been published. Given the information from the literature and presented herein, we suggest that genetic counseling for cases of PWS and 47,XXY should address these two conditions separately.

Genetic basis of familial Meniere's disease.

The genetic basis of familial Meniere's disease (MD) is unclear. We present a genetic investigation of six individuals in two families with familial MD. Linkage analysis was performed using polymorphic DNA markers linked to the human leukocyte antigen (HLA) locus that map to chromosome 6p. We have demonstrated the presence of anticipation in successive generations and the absence of HLA association. This is the second report of anticipation in familial MD in the literature, and it suggests that efforts should be directed toward finding a trinucleotide expansion as a possible genetic lesion in this uncommon disorder.