The Genetic Link welcomes guest blog authors. This guest submission is from Megan P. Hitchins, PhD. Medical Epigenetics Laboratory, Lowy Cancer Research Centre, University of New South Wales, Sydney, Australia. We thank Dr. Hitchins for her submission and welcome her as a guest author. I hope you enjoy this article.
A host of familial cancer syndromes have been described in which several members of the same family develop cancer at a young age due to an inherited genetic susceptibility. It has been well established that germline mutations in the DNA sequence of genes that are protective against cancer, including tumour suppressor and DNA repair genes, are the culprit in most familial cases of cancer. Because they are inherited, these germline mutations are present in every cell of the body from conception into adulthood, knocking out one of the two copies of the protective gene. They confer a high risk of cancer development at a young age, although the cancer itself arises when the remaining normally-functioning copy of the gene is knocked out in susceptible tissues due to contributing environmental conditions, taking with it the last remnants of protection it once afforded against cancer. However, for a number of individuals with young-onset cancer, as well as entire families, the inherited defect remains unidentified, which complicates genetic counselling and clinical management of family members. Lynch syndrome is the most common of all family cancer syndromes, in which patients develop a range of cancers, the most frequent of which are colorectal and uterine cancers. Lynch syndrome is usually caused by germline mutations within one of the four genes that encode the mismatch repair system, most commonly MLH1 or MSH2. Loss of protection from the mismatch repair system results in the accumulation of mutations during cell division, and ultimately, cancer ensues. However, in about a third of Lynch syndrome patients, standard genetic screening fails to identify any pathogenic sequence change within the mismatch repair genes that might be responsible for their disease.
The focus of the Medical Epigenetics Laboratory at the Lowy Cancer Research Centre in Sydney, Australia, led by Megan Hitchins PhD, is to determine the role of a new type of defect, termed a "constitutional epimutation" in predisposing to young-onset cancer syndromes. The first case of a "constitutional epimutation" was reported for the MLH1 gene in a patient with Lynch syndrome in 2002.1 This represents a new epigenetic mechanism of cancer susceptibility, in which the gene's promoter (the equivalent of its engine) is clogged with the chemical methylation, causing the affected copy to be switched off, even though its DNA code is entirely normal. Thus, "epimutation" refers to the nature of the defect, which occurs over and above the context of DNA sequence, whilst "constitutional" denotes the intrinsic presence of this epigenetic defect in normal tissues. Cancer similarly develops after the active copy of the gene is lost in the vulnerable tissues, giving rise to the same clinical profile as carriers of conventional sequence mutations of MLH1. However, one of the key differences between a constitutional epimutation and a sequence mutation is that the gene methylation can be unstable and sometimes show a "mosaic" pattern, that is, it may be present in a patchwork of some cell or tissue-types, whilst absent in others. The level of methylation in the body may even vary during the course of the carrier's life-time. This facet of epimutations adds to the challenge of identifying those who carry them, since their identifying factor - the presence of methylation - may not necessarily be detectable in DNA extracted from a single source, typically peripheral blood. A further complicating factor is that unlike germline mutations that demonstrate classic Mendelian patterns of inheritance, transmission of epimutations from one generation to the next is unpredictable. This non-Mendelian inheritance is attributable to the fact that methylation is stripped away during the reproductive life-cycle, and so epimutations tend to be reversed between generations and may, or may not, be re-imposed after fertilization in the developing fetus. Indeed, it is likely that the degree of methylation mosaicism witnessed in carriers reflects the stage of embryogenesis during which the methylation was established in them. Therefore, when it comes to screening for epimutations, for instance in the family members of a cancer patient found to carry one, it is prudent to test the DNA extracted from more than one tissue-type. Otherwise, it begs the question of whether a negative test means those relatives are safe from cancer as non-carriers, or whether it is just didn't show up in the tissue tested due to the mosaic nature of methylation. To minimise the possibility of failing to detect an epimutation, we have adopted the approach of testing DNA extracted from different tissue sources, namely blood, hair bulbs, buccal swabs, and thanks to the Oragene kits, saliva. In fact, since saliva is originally derived from the same embryonic cell lineage as the colon, it may even provide a more accurate representation of the epigenetic changes that have occurred in the colon than blood. In our recent article in the International Journal of Cancer,2 we show for the first time that in one case, Patient YT, who developed colon cancer at the age of just 18 years due to a constitutional MLH1 epimutation, that methylation was present in his saliva, as per other normal cells (Figure 1a). Thus his saliva showed a consistent pattern with other sources of tissue, indicating a severe soma-wide epimutation (Figure b). Furthermore, using the saliva RNA extraction kit, we were also able to show that the methylated copy of the gene was completely switched off (Figure 1c). Interestingly, screening of his parents showed that neither of them had any detectable methylation, despite screening multiple tissues including their saliva, and so we could say with confidence that the epimutation had arisen spontaneously in their cancer-affected son, but that they themselves were not at an elevated risk of developing cancer from the same type of defect.
Figure 1. Constitutional MLH1 epimutation in "Patient YT"
A: Schematic overview comparing a constitutional epimutation of MLH1, as found in Patient YT, with the normal gene of a healthy individual. Rectangles denote the two copies of the gene and its mRNA product. Methylation of the promoter is depicted by black lollipops. Gene activity is denoted by a waved arrow. The common c.655A>G SNP within the protein-code portion of the MLH1 gene is shown, which enables the two copies of the gene to be distinguished from one another, allowing the activity of the two alleles to be traced in the mRNA. B: Allelic patterns of MLH1 methylation showed methylation was widespread in normal somatic tissues, including saliva collected using the Oragene kit. The "beads of string" represent individual strands of DNA from the MLH1 promoter with dots showing positions of methylation present (black) or absent (white) at CG sites within the DNA sequence, at which methylation is capable of binding. C: Quantification by pyrosequencing of the two copies of the MLH1 gene in the genomic DNA and mRNA samples derived from saliva collected using the Oragene kits. The yellow-shaded region shows the peaks representing each allele at the SNP site. Two peaks showing equal levels of the ‘A' and ‘G' alleles in his saliva DNA show the patient is heterozygous for the benign c.655A>G SNP, allowing the two copies of to be differentiated. However, in his saliva mRNA, only the ‘G' allele was detected, indicating that the ‘A' allele has been switched off by the promoter methylation further upstream.
Our laboratory now routinely collects saliva using the Oragene kits from patients with colon cancer who we suspect may have been caused by intrinsic epigenetic changes, since we believe methylation may be elevated and hence more easily detected in this source of DNA than in blood. Furthermore, we are finding that our rate of compliance in providing samples for diagnostic or research purposes has increased. Firstly, those who are elderly, sick or have an aversion to needles are more willing to provide a sample of saliva than blood for DNA or RNA extraction. Secondly, in our research endeavouring to unravel the inheritance pattern of constitutional MLH1 epimutations, we rely on the voluntary contribution of specimens from the asymptomatic relatives of cancer patients who carry this defect. Relatives are happier to provide a specimen of saliva at their own convenience and in the comfort of their own home, than in visiting their clinician or pathology laboratory to provide a blood sample. Following contact by phone with the genetic counsellor or research nurse, they simply place their saliva sample in the stamped addressed envelope we provide and return it to the laboratory by post, along with their signed consent form approving the inclusion of their sample in our research study. To this end, the Oragene saliva kits have greatly facilitated our research from a logistical perspective, whilst also providing a new dimension for testing different tissue-types for epigenetic changes that may vary from one tissue source to another.
References.
- 1. Gazzoli, I., Loda, M., Garber, J., Syngal, S. & Kolodner, R.D. A hereditary nonpolyposis colorectal carcinoma case associated with hypermethylation of the MLH1 gene in normal tissue and loss of heterozygosity of the unmethylated allele in the resulting microsatellite instability-high tumor. Cancer Res 62, 3925-8 (2002).
- 2. Goel A, Nguyen T-P, Hon-Chiu E Leung H-CE, Nagasaka T, Rhees J, Hotchkiss E, Arnold M, Banerji P, Koi M, Kwok C-T, Packham D, Lipton L, Boland CR, Ward RL, Hitchins MP. De novo constitutional MLH1 epimutations confer early-onset colorectal cancer in two new sporadic Lynch syndrome cases, with derivation of the epimutation on the paternal allele in one. International Journal of Cancer 2010, in press.
Recent statistics suggest cancer mortality rates are declining due to better prevention, early detection methods and improved treatments, yet so much remains to be done. With cutting-edge research continually pushing the boundaries of science and discovery, it is not surprising that an increasing number of cancer researchers are turning to the newest tool in the DNA collection toolbox - DNA from saliva.
Saliva has traditionally been overlooked as a source of DNA for cancer research but Oragene•DNA has changed that. What makes saliva-based DNA collection with Oragene•DNA so important to cancer research? Saliva provides a non-invasive means of collecting high quality and quantity DNA that is stable at room temperature, which makes samples easy to collect, store and ship. With geographically dispersed subjects, scientists can even mail Oragene•DNA kits to participants, who follow the directions, and return the saliva samples to the laboratory for analysis using standard mail service. Cancer research groups around the world are now focusing on the potential of this common bodily fluid for detecting the genetic link to the disease and studying genes without the need for a blood draw.
Now that researchers trust that DNA from saliva using Oragene is equivalent to DNA from blood, the door is open to populations they would normally not have access to. Collecting blood is very invasive and not a practical procedure for children or individuals that can't give blood for religious or medical reasons or for those who do not have access to a collection center. Compliance rates improve with saliva resulting in the collection of more samples.
Breast cancer researchers at the University of Arkansas for Medical Sciences (UAMS) are hoping to answer some important questions with DNA from saliva. They are building a repository aimed at studying breast cancer risk and treatment. Their goal is to collect 40,000 samples over a five year period. They want to learn why one individual is affected by breast cancer but another is not. Or why two women with the same disease respond differently to the same treatment. What inherited factors contribute to the disease? What environmental factors contribute to breast cancer? How do inherited and environmental factors interact to cause the disease?
The researchers decided to use Oragene•DNA because saliva-based collection offered them a non-invasive, easy-to-use and reliable method to collect the large number of DNA samples they need. The kit stabilizes DNA at room temperature until it can be analyzed, which eliminates storage and logistical issues. In addition, Oragene•DNA provides the high quality, high quantity DNA required for establishing the bio-repository. The reliability and ease-of-use also enabled on site event-based collections bolstering the donor group by thousands in a single day.
In another example, researchers at Inserm (I'Institut national de la santé et de la recherché medicale) in France are conducting a study on thyroid cancer using Oragene•DNA. The primary goal of the project is to better understand the risk factors associated with the development of thyroid cancer. The project will establish a DNA bank to permit future studies of candidate genes linked with thyroid cancer and to study gene-environment interactions. The team evaluated the possibility of collecting blood samples, buccal swabs or Oragene•DNA. After evaluating all options, they decided to use Oragene•DNA as it was the only method that allowed them to overcome their main challenge of maximizing compliance rates for geographically dispersed participants.
So, is saliva-based DNA collection the newest tool in the fight against cancer? Enabled by Oragene•DNA, the answer is a definitive ‘yes'. Cancer research with saliva DNA has definitely come of age.
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Every day, it seems, scientists learn something new about how our genes work. One fascinating area of research involves understanding the role of our genes in the initiation, progression and treatment of diseases; such as cancer. Understanding cancer on a molecular and genetic level makes for good science and good medicine. We understand that all cancers are not created equally. From the moment you are conceived, your genes may increase your susceptibility to developing certain cancers or, later on, your environmental exposures or other factors may cause changes in your genes that cause cancer to develop. Cancer is not one disease, but many, adding to the complexity and breadth of studies.
Research is the best way to fully understand the mechanics of this disease and ultimately develop better strategies to combat it. Scientists and clinicians alike are constantly working to learn more about the role of genetics in cancers so they can improve treatment options and health outcomes for patients. The study of genes and cancer all share a fundamental requirement - they all need DNA.
The collection and analysis of DNA from blood and tissue have long been considered the golden standards in cancer research studies. Obtaining high quality genomic DNA is critical for studies that aim to evaluate the role of genetic factors in cancer. However, cancer research studies often require very large numbers of samples from a dispersed population and non-invasive methods for DNA collection. Saliva samples, which are painless for the donor and relatively easily collected, are quickly becoming the preferred choice.
Oragene•DNA is the product of choice for many cancer researchers who require a safer, simpler mechanism for collecting genetic samples than the traditional method of blood collection. This method of DNA collection is highly desirable in certain patient groups (e.g., children, those fearful of venipuncture, geographically dispersed study populations, or as a back up source of DNA in studies that collect blood).
Oragene•DNA kits are being used today in a wide variety of cancer studies including those investigating candidate genes and inherited risk family studies. Oragene•DNA is well suited for cancer research applications including:
Genetic Research: to identify genetic targets for therapy or diagnostics;
Genetic Screening: to help determine who is at high risk of developing cancer and who would have the better prognosis;
Pharmacogenomics: to determine the influence of genetics on treatment choice and disease prognosis.
One example of the type of cancer research that is benefiting from non-invasive DNA sample collection is breast cancer. One such research study was published in 2009: Christine B. Ambrosone, Gregory L. Ciupak, Elisa V. Bandera, et al., "Conducting Molecular Epidemiological Research in the Age of HIPAA: A Multi-Institutional Case-Control Study of Breast Cancer in African-American and European-American Women," Journal of Oncology, vol. 2009, Article ID 871250, 15 pages, 2009. doi:10.1155/2009/871250.
The authors of this research conducted a case-control study with the goal of recruiting 1200 African American and 1200 European American women with breast cancer and an equal number of controls in order to evaluate numerous risk factors for early/aggressive breast cancer and to evaluate the distribution of these risk factors within and across racial/ethnic groups. They initially collected blood samples which were processed and stored in the laboratory at the Mount Sinai School of Medicine. In 2007, to reduce costs and to facilitate participation, they transitioned to collection of saliva using Oragene•DNA kits for DNA extraction. The authors state: "These collection kits yield large quantities of high quality DNA, comparable to that obtained from whole blood."
If you'd like more details on how the researchers benefitted from non-invasive DNA collection in this breast cancer research study, download the full copy here.
At DNA Genotek, we're committed to creating educational resources for researchers involved in cancer genetics and to facilitate the sharing of best practices for DNA collection. If you have information to share or would like to contribute to this blog, please send us your suggestions and ideas.
We'll be posting more articles on saliva DNA collection methods for cancer research in the coming weeks. Don't forget, The Genetic Link has subscription options; you can follow by email or RSS feed.