Family Studies for Classification of Variants of Uncertain Classification: Current Laboratory Clinical Practice and a New Web-Based Educational Tool

Multi-gene cancer panels often identify variants of uncertain clinical significance (VUS) that pose a challenge to health care providers in managing a patient’s cancer risk. Family segregation analysis can yield powerful data to re-classify a VUS (as either benign or pathogenic). However, financial and personnel resources to coordinate these studies are limited. In an informal assessment we found that family studies for variant classification are done by most clinical genetics laboratories that offer hereditary cancer panel testing. The process for family studies differs substantially across laboratories. One near universal limitation is that families usually have too few individuals for an informative co-segregation analysis. A unique and potential resource-saving approach is to engage patients and their families in expanding their own pedigrees for segregation analysis of their VUS. We describe a novel public educational tool (FindMyVariant.org) designed to inform patients and genetic counselors about strategies to improve the probability of variant classification using familial segregation. While the web tool is designed to be useful for any gene, the project was primarily focused on VUS’s returned in cancer risk genes. FindMyVariant.org is a resource for genetic providers to offer motivated families who are willing to gather information about their family relationships and history. Working alongside clinical or research genetic laboratories, the information they collect may help reclassify their VUS using segregation analysis.


Introduction
Panel gene sequencing has become an increasingly important strategy for evaluating inherited disease risk. Gene panels are now available for the work-up of conditions such as aortopathy, arrhythmia, cancer syndromes, cardiomyopathy, epilepsy, immunodeficiency, mitochondrial disorders, and Xlinked intellectual disability (Chambers et al. 2016;Falk et al. 2012;Green et al. 2015;Hunter et al. 2014;Pritchard et al. 2014;Walsh et al. 2011). Expanded gene panels are more sensitive than single gene testing, and are often more cost effective than sequential testing, leading to additional diagnostic and prevention opportunities (Gallego et al. 2015). However, these panels have the caveat of also identifying rare variants of uncertain clinical significance (VUS) in a higher proportion of patients compared to single gene testing alone (Cragun et al. 2014;Kurian et al. 2014;Maron et al. 2012;Maxwell et al. 2014;Tung et al. 2014). A VUS typically has some characteristics or associated data indicating the variant may be deleterious, but not enough information to definitively classify it as disease causing, or pathogenic. Millions of such variants are present at low frequencies in the population (Lek et al. 2015).
The finding of a VUS can be problematic for patients and clinicians working in a clinical genetics setting (Culver et al. 2013;O'Neill et al. 2006;van Dijk et al. 2006;Vos et al. 2008). Although definitive re-classification of a VUS as pathogenic or benign may eventually occur, the timeline is typically many years, and may be indefinite for rare VUS, especially if the disease is uncommon (Lindor et al. 2013;Murray et al. 2011;O'Neill et al. 2009). In-silico or functional research studies usually cannot resolve the clinical significance of a variant (Katsonis et al. 2014;Lindor et al. 2012;Moghadasi et al. 2013;Schulz et al. 2015). While family segregation studies have the potential to yield powerful data to classify variants, many challenges emerge that limit this potential. The resources needed to identify distant family members and build the large pedigrees necessary to have enough information for informative segregation analysis are inadequate. (Goldgar et al. 2008;Mohammadi et al. 2009). While research studies dedicated to investigating rare variants in families could impact individual families, current research resources and funding are limited and unlikely to have an influential impact on a large number of families.
Most clinical genetics laboratories that offer multigene cancer panel testing do family studies for variant classification; however, practices and policies vary. Recognizing the need to assess the current landscape of family studies across clinical labs, we performed an informal assessment of 12 clinical laboratories located in the United States that offer cancer multigene panel testing using Next Generation Sequencing to determine their current VUS classification activities and procedures.
A major limitation to family studies for VUS reclassification is that the size of the family reported to genetics providers in clinical visits often includes only three generations of relatives and is too small to enable VUS classification (Eggington et al. 2013;Shirts et al. 2013). A majority of patients will have sufficient relatives to reclassify VUS within their first and second cousins (B.H. Shirts and Rosenthal 2015). However, explanation of the details of familial co-segregation studies may be beyond the scope of what could be expected of a genetics provider in routine clinical care.
In response to needs of patients with VUS reports and genetics providers who may be interested in family studies to classify VUS, we developed a novel public educational tool designed to inform patients and genetic counselors about strategies to improve the probability of VUS re-classification using familial segregation. The website will also be a resource for patients, genetic counselors, and other health professionals seeking clinical laboratories and research studies that perform family analysis for VUS classification. This web resource can be found at FindMyVariant.org.

Assessment of Genetics Laboratories Offering Family Analysis Services for VUS Classification
We contacted 12 commercial clinical genetics laboratories by telephone and email and asked about current VUS reclassification activities, policies, and procedures between July and November 2015. Our questions were designed to include any VUS re-classification services as follow up on VUS findings in any tested gene where a VUS has been identified. Although these services benefit families that are able to reclassify their VUS, as accurate classification enables appropriate clinical care, (Murray et al. 2011), there is no established standard of care for VUS re-classification using family studies. Our goal was not to formally compare laboratories across specific metrics, but rather, to learn the current landscape of VUS re-classification practices.
We initially asked laboratories several broad questions: Is there a family studies program in place? What are the criteria and/or policy for eligibility? And what is the process for enrollment and sample collection? Based on responses we asked for clarification. We re-contacted several laboratories for clarification and to gather additional information. Representatives of each laboratory were given the opportunity to review final content for accuracy, although not all laboratories responded to our queries. Information about Myriad Genetics was gathered from the BMyVision^Variant Classification Program website. In addition to the laboratories listed in Table 1, we contacted Quest and Prevention Genetics, but these laboratories had no description of family studies on their respective web sites and did not respond to our phone queries. Because of the lack of community standards for VUS classification activities and the wide variety of practices, our assessment was not designed as a formal survey.

Assessment Results
There are similarities and differences across the labs (See Table 1). Several commercial genetics laboratories (e.g., Ambry, GeneDx, Invitae) have family studies programs in place with well-developed inclusion/exclusion criteria and/or policies. For example, one lab excludes certain genes from their family studies program, such as low-moderate penetrance cancer genes like ATM, BARD1, BRIP1, CHEK2, MRE11A, NBN, RAD50, RAD51C, and RAD51D. Other laboratories tend to review family studies candidates on a caseby-case basis (e.g., ARUP laboratories, GeneDx, the University of Washington). Nearly all laboratories offer no laboratory fee for familial single-site testing for relatives that would inform segregation analysis for at least a subset of genes and carefully selected families. All laboratories that offer any form of family studies reported that the program began the same time they started offering clinical cancer gene testing. When asked how many families have been eligible for family studies and what the reclassification Bsuccess^rate was, each laboratory reported no mechanism for keeping track of how many patients were eligible, offered participation, or ultimately how many variants were reclassified due to successful co-segregation analysis via family studies. One interviewee explained that although there were many different ways and reasons a VUS could be reclassified, the reason *Of the twelve commercial clinical labs we surveyed, two labs, Prevention Genetics and Quest, did not respond to our inquiry why it was being reclassified, such as family study information, wasn't being tracked. In general, offering family studies was a common but not necessarily uniform practice for these clinical laboratories that perform genetic testing for hereditary cancer risk. Some of the most significant differences between laboratories are in the process for family studies. Some clinical laboratories work with patients and genetic counselors encouraging individuals to reach out to known family members and identify additional relatives in order to reclassify variants. For most clinical laboratories, however, family studies were only offered in selected situations where VUS reclassification is most likely to be possible or only for selected genes. Some laboratories offer family segregation studies to any interested family on a feefor-service basis.

Motivation to Develop General Online Resources for Family Based VUS Classification
As can be seen in Table 1, many clinical genetics laboratories currently offer family studies programs. These programs are diverse in how they operate and many place the onus on genetics providers to order testing and to help patients identify relatives and obtain biological specimens. Neither genetic counseling time nor the laboratory resources used for family study activities are reimbursed by insurance companies. Limited genetics provider resources and laboratory opportunities make it challenging for many willing patients to participate in successful VUS reclassification efforts.
While advertising the availability of family testing services, clinical laboratories simultaneously have mechanisms to control and restrict patient access to family segregation studies. Commercial laboratories may feel obliged to offer family based VUS reclassification services in limited instances because it is expected by genetics providers; however, there is little incentive to assist patients in contacting the large number of relatives necessary to reclassify VUS or to provide broad access to education about family studies for VUS classification as testing large numbers of relatives can be costly for laboratories. More importantly, the time and effort needed to work with patients and their relatives to build the 4-5 generation pedigrees that are usually necessary to classify VUS would be administratively challenging and prohibitively expensive.
A potential solution is to engage patients and their relatives in expanding their own pedigree. Although there are limited provider and laboratory resources for personally working with and educating patients about pedigree building for family studies, online genealogy resources and social networking may facilitate patients identifying and contacting their relatives themselves with minimal personal guidance. This observation sparked our interest in developing an educational resource for patient-driven family history building for the purpose of VUS classification.

FindMyVariant.org, a New Resource for VUS Family Studies
We have developed a website that contains a patient-driven VUS reclassification toolkit. The mains goals of the toolkit are to: help individuals and families understand medical uncertainty about rare variants; educate individuals and families about ways to find more information about the variants that are unique to their family; provide resources for individuals who want to connect with their families to increase understanding about their genetic variants, and finally, provide resources for individuals who want to connect with clinical laboratories or research studies to learn more about their VUS.
The toolkit walks individuals step-by-step through the process of gathering the family specific segregation information required to reclassify any VUS that has been identified in clinical testing. This toolkit explains the variant reclassification process, teaches patients how to identify relatives who may also carry the VUS, (i.e. informative relatives), and provides aids for contacting relatives and describing the need to involve them in VUS classification. We sought insight from clinical genetic counselors, molecular genetic pathologists, bioethicists, and clinical geneticists to develop content for the website, and we worked alongside an experienced web developer to design the website in a patient-and userfriendly manner.
The content of the website is designed and organized around eight modules or Bsteps^that a patient may encounter when gathering family medical and variant data ( Table 2). The modules walk the patient through each step and include graphics and examples. Screenshots of the home page and of different modules are presented in Figs. 1, 2, 3 and 4. One module lists several online genealogy and social networking tools available to facilitate patients identifying and contacting their relatives, and describes how these might be used in the context of family history building for variant classification (see Fig. 3). In addition to this content, the website contains two examples that describe the experience of hypothetical individuals going through the process of attempting to reclassify the familial VUS following the tools offered in the website (see Fig. 4).
BAmelia's family^is an example of a woman with breast cancer who has a VUS in BRCA1. It includes descriptions of Amelia talking to her family, trying to obtain tumor tissue from a deceased ancestor, and using social media to find distant relatives. Ultimately, her VUS is tracking with cancer in her family and can be reclassified as pathogenic, causing increased cancer risk in relatives who carry the same variant. Similarly, BCharlie's family^is an example of a man with Contact your living parents and siblings Explains how to collect, record, and confirm basic facts about close relatives.

Using genealogy to identify ancestors who might have your variant
Talking with living relatives to find your ancestors Teaches how to gather information from living relatives about deceased ancestors.
Using online genealogy tools to find your ancestors Lists several genealogy resources including major family history websites with a very brief description of how to use these resources.
Obtaining tumors from deceased ancestors for testing Introduces the potential option of getting tissue from pathology samples from deceased ancestors and briefly describes this process.

Finding and Connecting with distant relatives that might have your variant
Start with the people you know Explains that the best way to find distant relatives is by starting with people that you already know Talking with living relatives to find descendants of your ancestors Describes how to talk to known relatives about descendants of a common ancestor, including how to ask for contact information of distant relatives.
Using online social networking sites to find descendants of your ancestors Provides examples of how to use social networking sits, like Facebook, to identify distant relatives.

Don't forget low-tech
Lists several public search engines used for finding information about relatives and a variety of people search tools.

Laboratory Testing and asking relatives about participation in VUS classification
Finding a clinical Laboratory to Test Samples and Classify Your Variant Lists family study resources offered by several clinical laboratories in the United States.

Finding a Research Laboratory to Test
Samples and Classify Your Variant Lists contact information for research studies performing family studies for VUS reclassification. colon cancer and a VUS in APC that might be associated with attenuated familial adenomatous polyposis. This example includes descriptions of a patient talking to his family, uncovering information about his great-grandparents, and discovering a branch of his family tree of which he was not aware. The example concludes with Charlie learning that the genetic variant he carries does not cause cancer risk and is reclassified as likely benign. The goal of these examples is to mimic as realistically as possible some of the smaller successes and challenges a motivated patient could expect to come across when attempting to gather all the information that is needed to successfully classify a variant using co-segregation analysis in their own family. Further, they demonstrate the two different outcomes that can be expected upon successful completion of family studies: in Amelia's family, the VUS was reclassified to pathogenic, while in Charlie's family, his VUS was reclassified as likely benign (See Fig. 4, Table 2).
One specific challenge to patients with VUS that are interested in family studies to learn more about their VUS, is the difficulty in obtaining laboratory services to genotype and classify their variants. As illustrated in Table 1, each laboratory has different processes and criteria for eligibility. Laboratories also have different variant classification criteria and may draw different conclusions from the same family data (Yorczyk et al. 2015). Although, the BLaboratory Testing and Asking Relatives About Participation in VUS Classification^module of the web tool lists clinical laboratories and research investigations involved in family studies and provides links to relevant information, we could not outline next steps for interacting with clinical and research laboratories because there is no uniform process. This is a stage in family studies for variant classification where the patient will necessarily need to contact the testing laboratory for guidance about how to proceed.

Discussion
The issue of large numbers of patients with variants of uncertain significance is already a problem for medical genetics practice (Culver et al. 2013;O'Neill et al. 2009;Lindor et al. 2013), and may become greater as large-scale sequencing enters clinical care. It is estimated that each individual has hundreds of rare, family-specific variants that might be classified as VUS. Interpreting VUS in clinical genetics creates challenges for health care providers, and their patients. Despite the potential of family studies for VUS reclassification, the resources available and offered to patients are limited.
We surveyed 12 laboratories offering NGS panels for cancer genes that perform family studies for genetic variant classification and provide a summary of eligibility criteria and processes where these details are defined by the testing laboratories reviewed. While not designed to be a formal survey of laboratory practices, we hope this information is a useful resource for genetic counselors and other health professionals as they help patients found to carry a VUS to understand potential next steps for resolving its clinical significance.
While family studies have the potential to yield powerful data to reclassify variants, the time and effort required to build pedigrees adequately for variant classification limit this potential. We developed an educational website containing a patient-friendly toolkit to help aid patients in working with research and clinical laboratories to reclassify their own VUS. The modules were developed to walk patients step by step through the process and address some of the realistic challenges that might come up along the way. Recognizing that each patient and family has it's own complexity and challenges, we offered two examples of patients ( Fig. 4: Amelia and Charlie, see screenshot) from families suspicious for hereditary breast and hereditary colorectal cancers, respectively, as representative examples.
While the website was developed with the above benefits in mind, we recognize that this is a first attempt at designing a patient-friendly toolkit for variant reclassification and consequently, has limitations. At this time, the toolkit is designed for English speaking patients. Further, while we have attempted to build resources suitable for most patient education levels, we realize that some of the concepts and content may be beyond the comprehension level of some patients or their relatives. Because the website conveys complex terms in a simplified level, it may be easier for patients of all educational levels to use. Each patient's family is different and subsequently, the number of individuals needed to reclassify a VUS will be different for each family, and based on the gene and rarity of the disease. There are potential barriers to patients having access to the Fig. 1 Screenshot of the home page for FindMyVariant.org, a public website. Patients can click on the Bget started^prompt and will be introduced to a number of different modules (designed in order, as seen in Table 3) to walk them through the process of gathering family information to help them classify their variant  Screenshot with an example of another educational module found at FindMyVariant.org. This module walks a patient through the process of using online genealogy and social networking tools and resources to find and connect with relatives website, either because their ordering health care provider is not aware that it exists, or they or their relatives may not have internet or computer resources necessary to access the website. Privacy and data security concerns prevented us from including interactive pedigree building at this time; we are investigating this as a possibility in future versions.
As more patients use the website, other challenges that we did not anticipate are likely to be identified. Although reclassification is theoretically possible for almost all families given enough effort in pedigree building, practicalities may prevent many individuals from being able to use family studies to reclassify their variants such as individuals who were adopted, families who used assisted reproductive technologies utilizing donor gametes, or individuals who have limited knowledge or contact with relatives. Further, it is possible that only highly motivated patients will be willing to make the effort to grow their family histories to the point where they are able to classify their variants. Each laboratory we interviewed uses different rubrics for identifying individuals that qualify for family studies and for reclassifying VUS through family studies. Laboratories will undoubtedly respond differently to patient efforts to grow their pedigrees for successful co-segregation analysis. There are still many unanswered questions about VUS classification. Few laboratories report keeping quality records of the success rate of family studies, so future studies will be needed to explore patient satisfaction with VUS classification activities and outcome metrics of these efforts.

Conclusion
In the current paradigm of multi-gene panel testing, the return of a result with one or more VUS's is a challenge faced by genetic and other health care providers and ultimately, their patients and families. Many laboratories are involved in conducting family studies for variant reclassification with diverse protocols and eligibility criteria.
Engaged patients who are empowered to overcome this barrier to VUS reclassification may make it possible for variant reclassification to shift from that of a research activity, to a clinical activity enabling individualized genomic medicine. In addition to enhancing understanding of rare variants encountered in clinical testing, this approach could provide a significant benefit to families faced with VUS findings. We have described a web tool that responds to this need, enabling patients to take charge of their genetic information in the age of genomic medicine.
Acknowledgments The authors would like to thank Stephanie Malia Fullerton and Mary-Claire King who provided feedback about web content. The authors would also like to thank the clinical laboratory contacts that provided information about VUS classification activities at their respective laboratories.
Funding Brian Shirts is a Damon Runyon-Rachleff Innovator supported in part by the Damon Runyon Cancer Research Foundation (DRR-33-15). Additional funding for this project was provided by the National Human Genome Research Institute (R21 HG008513) and the Fred Hutch /University of Washington Cancer Consortium Cancer Center Support Grant from the National Cancer Institute (5P30 CA015704-39) to Brian Shirts. Laura Amendola is funded by the National Human Genome Research Institute and National Cancer Institute grant U01 HG006507.

Compliance with Ethical Standards
Conflicts of Interest All authors declare they have no financial conflicts of interest.
Human Studies and Informed Consent This research was determined not to be human subjects or animal research using University of Washington Human Subjects Division protocols.
Open Access This article is distributed under the terms of the Creative Comm ons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Fig. 4 Screenshot illustrating one of two Bfamily examples^on the website. Amelia is a patient who is eager to classify her BRCA1 VUS; this example demonstrates Amelia's journey using different modules to help her connect with relatives and gather the necessary information need to ultimately reclassify her VUS to a pathogenic variant, conferring cancer risks for both her and her relatives also carrying the BRCA1 variant