23andMe® Personal Genome Service® (PGS®) Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants)

K223597 · 23AndMe, Inc. · QAZ · Aug 31, 2023 · Medical Genetics

Device Facts

• Record ID: K223597
• Device Name: 23andMe® Personal Genome Service® (PGS®) Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants)
• Applicant: 23AndMe, Inc.
• Product Code: QAZ · Medical Genetics
• Decision Date: Aug 31, 2023
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.6090
• Device Class: Class 2
• Attributes: Software as a Medical Device, PCCP

Indications for Use

The 23andMe Personal Genome Service (PGS) uses qualitative genotyping to detect select clinically relevant variants in genomic DNA isolated from human saliva collected from individuals ≥18 years with the Oragene Dx model OGD500.001 for the purpose of reporting and interpreting genetic health risks, including the 23andMe PGS Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants).

Device Story

Device uses qualitative genotyping to detect 44 specific BRCA1/BRCA2 variants in human saliva. Saliva collected via Oragene-Dx device; DNA isolated and processed using multiplex microarray technology (Illumina BeadChip v5). DNA fragmented, hybridized to SNP-specific primers, extended with hapten-labeled nucleotides, and detected via fluorescently labeled antibodies. Tecan Evo and Illumina iScan systems perform extraction, processing, and scanning. Raw data analyzed by GenomeStudio and 23andMe proprietary Coregen software to determine genotypes. Results integrated into personalized reports for OTC user access. Reports provide information on detected variants and associated cancer risks (breast, ovarian, prostate, pancreatic). Device includes a Pre-Determined Change Control Plan (PCCP) for adding future validated variants. Output informs user-healthcare provider discussions; does not diagnose disease or determine treatment. Benefits include providing genetic risk information to individuals for proactive health management.

Clinical Evidence

Bench testing only. Method comparison study (n=samples from database) vs. bidirectional Sanger sequencing demonstrated >99% PPA and NPA. Precision study (intra-assay, inter-lot, inter-instrument, inter-operator, inter-day, inter-lab) showed 100% correct genotype calls. Minimum DNA input study validated performance across 5-50 ng/μL range. Pathogenicity of variants supported by peer-reviewed literature and ENIGMA consortium criteria.

Technological Characteristics

Qualitative genetic test for SNP detection. Sample: genomic DNA from saliva. Method: bead array hybridization, primer extension, fluorescent antibody detection. Instrumentation: Illumina iScan, GenomeStudio. Software: Coregen. PCCP implemented for variant expansion. Materials: Oragene Dx OGD500.001 collection kit.

Indications for Use

Indicated for reporting 44 specific BRCA1 and BRCA2 variants in adults ≥18 years. Provides genetic information regarding increased risk for breast, ovarian, prostate, and pancreatic cancers. Not for diagnostic use, treatment determination, or overall cancer risk assessment. Over-the-counter use.

Regulatory Classification

Identification

A qualitative in vitro molecular diagnostic system used for the detection of select variants in specified cancer-related genes. The device is intended to be used on genomic DNA isolated from human specimens collected by the user. The results of the test provide users with a genetic health risk assessment for developing certain cancers. The test may not include all variants associated with a predisposition of developing cancer and is not intended to describe a person’s overall risk of developing any type of cancer nor to aid in determination of treatment or act as a substitute for recommended cancer screenings or appropriate follow-up. The device is for over-the-counter use.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) The labeling required under § 809.10 of this chapter and any pre-purchase page and test report generated, unless otherwise specified, must include: (i) An intended use that specifies in the indications for use the genetic variants detected by the test. The specific variants must be appropriately validated as described in paragraphs (b)(4)(xii) and (b)(4)(xiii) of this section. (ii) A section addressed to users with the following information: (A) A warning statement accurately disclosing the genetic coverage of the test in lay terms, including information on variants not queried by the test, and the proportion of pathogenic variants in the genes that the assay detects in a specific population as identified in paragraph (b)(1)(i) of this section. The warning statement must indicate that the test [does not/may not, as appropriate] detect all genetic variants related to the genetic disease, and that the absence of a variant tested does not rule out the presence of other genetic variants that may impact cancer risk. The warning statement must also include the relevant population for which the variants reported by the test are most relevant. (B) A limiting statement explaining that some people may feel anxious about getting genetic test health results. This is normal. If the potential user feels very anxious, such user should speak to his or her doctor or other healthcare professional prior to collection of a sample for testing. This test is not a substitute for visits to a doctor or other healthcare professional. Users should consult with their doctor or other healthcare professional if they have any questions or concerns about the results of their test or their current state of health. (C) A limiting statement that a user's ethnicity may affect whether the test is relevant for them and may also affect how their genetic health results are interpreted. (D) A warning statement that the test is not a substitute for visits to a healthcare professional for recommended screenings, and should not be used to determine any treatments or medical interventions. (E) A warning statement that the test does not diagnose cancer or any other health conditions and should not be used to make medical decisions. The warning statement must indicate that the results should be confirmed in a clinical setting before taking any medical action. (F) A limiting statement explaining that other companies offering a genetic risk test may be detecting different genetic variants for the same disease, so the user may get different results using a test from a different company. (G) If applicable, a limiting statement that states the test does not test for variants in other genes linked to hereditary cancer. (H) A limiting statement explaining that this test does not account for non-genetic factors and that other factors such as environmental and lifestyle risk factors may affect the risk of developing a given disease. (I) Information to a potential purchaser or actual test report recipient about how to obtain access to a board-certified clinical molecular geneticist or equivalent to assist in pre- and post-test counseling. (J) A limiting statement explaining that this test is not intended to tell you anything about your current state of health, or be used to make medical decisions, including whether or not you should take a medication or how much of a medication you should take. (K) A limiting statement explaining that the laboratory may not be able to process a sample, and a description of the next steps to be taken by the manufacturer and/or the customer, as applicable. (iii) A section in the labeling required under § 809.10 of this chapter and any test report generated that is for healthcare professionals who may receive the test results from their patients with the following information: (A) A limiting statement explaining that this test is not intended to diagnose a disease, determine medical treatment or other medical intervention, or tell the user anything about their current state of health. (B) A limiting statement explaining that this test is intended to provide users with their genetic information to inform health-related lifestyle decisions and conversations with their doctor or other healthcare professional. (C) A limiting statement explaining that any diagnostic or treatment decisions should be based on confirmatory prescription testing and/or other information that is determined to be appropriate for the patient ( *e.g.,* additional clinical testing and other risk factors that may affect individual risk and health care).(2) The genetic test must use a sample collection device that is FDA-cleared, -approved, or -classified as 510(k) exempt, with an indication for in vitro diagnostic use in over-the-counter DNA testing. (3) The device's labeling must include a hyperlink to the manufacturer's public website where the manufacturer must make the information identified in paragraph (b)(3) of this section publicly available. The manufacturer's home page, as well as the primary part of the manufacturer's website that discusses the device, must provide a hyperlink to the web page containing this information and must allow unrestricted viewing access. If the device can be purchased from the website or testing using the device can be ordered from the website, the same information must be found on the web page for ordering the device or provided in a publicly accessible hyperlink on the web page for ordering the device. Any changes to the device that could significantly affect safety or effectiveness would require new data or information in support of such changes, which must also be posted on the manufacturer's website. The information must include: (i) An index of the material being provided to meet the requirements in paragraph (b)(3) of this section and its location. (ii) Technical information about the device, as specified in paragraph (b)(4) of this section. (iii) A section that highlights summary information that allows the user to understand how the test works and how to interpret the results of the test. This section must, at a minimum, be written in plain language understandable to a lay user and include: (A) Consistent explanations of the risk of disease associated with all variants included in the test, variants not included in the test, and specific considerations by ethnicity. If there are different categories of risk, the manufacturer must provide literature references and/or data that support the different risk categories. If there will be multiple test reports and multiple variants, the risk categories must be defined similarly among them. For example, “increased risk” must be defined similarly between different test reports and different variant combinations. (B) Clear context for the user to understand the context in which the cited clinical performance data support the risk reported. This includes any risks that are influenced by ethnicity, age, gender, environment, and lifestyle choices. (C) Materials that explain the main concepts and terminology used in the test that include: ( *1* ) Definitions: scientific terms that are used in the test reports.( *2* ) Pre-purchase page: this page must contain information that informs the user about what information the test will provide. This includes variant information, the condition(s) or disease(s) associated with the variant(s), professional guideline recommendations for general genetic risk testing, the limitations associated with the test (*e.g.,* test does not detect all variants related to the disease), relevance of race/ethnicity, and any precautionary information about the test the user should be aware of before purchase. When the test reports the risk of a life-threatening or irreversibly debilitating disease or condition for which there are few or no options to prevent, treat, or cure the disease, a user opt-in page must be provided. This opt-in page must be provided for each disease type that falls into this category and must provide specific information relevant to each test result. The opt-in page must include:( *i* ) An option to accept or decline to receive this specific test result;( *ii* ) Specification of the risk involved if the user is found to have the specific genetic test result;( *iii* ) Summary of professional guidelines that recommend when genetic testing for the associated target condition is or is not recommended;( *iv* ) A recommendation to speak with a healthcare professional, genetic counselor, or equivalent professional before getting the results of the test;( *v* ) The implications of receiving a no variants detected result; and( *vi* ) The statement that the test does not diagnose cancer or any other health conditions and should not be used to make medical decisions. Results should be confirmed in a clinical setting before taking any medical action. Users should consult with a healthcare professional before taking any medical action.( *3* ) Frequently asked questions (FAQ) page: This page must provide information that is specific for each variant/disease pair that is reported. Information provided in this section must be scientifically valid and supported by corresponding peer-reviewed publications. The FAQ page must explain the health condition/disease being tested, the purpose of the test, the information the test will and will not provide, the relevance of race and ethnicity to the test results, information about the population to which the variants in the test is most applicable, the meaning of the result(s), other risk factors that contribute to disease, appropriate follow-up procedures, how the results of the test may affect the user's family, including children, and links to resources that provide additional information.(4) The device labeling must include a technical information section containing the following information: (i) Gene(s) and variant(s) the test detects using standardized nomenclature, Human Genome Organization (HUGO) nomenclature and coordinates, as well as Single Nucleotide Polymorphism Database (dbSNP) reference SNP numbers (rs#). (ii) A statement indicating that more than 1,000 variants in the BRCA1 and BRCA2 genes are known to increase cancer risk, as applicable. (iii) Scientifically established disease-risk association of each variant detected and reported by the test. This risk association information must include: (A) Genotype-phenotype information for the reported variants. (B) When available, a table of expected frequency in the general population and different ethnicities, and risks of developing the disease in relevant ethnic populations and the general population. (C) Information such as peer-reviewed published literature and/or professional guidelines used to determine what types and levels of evidence will distinguish whether the selected variants are reported as “are associated with increased risk” versus “may be associated with increased risk” of developing other cancers. All selected variants must be appropriately validated as required under paragraph (b)(1)(i) of this section. For selected variants reported as “are associated with increased risk,” the clinical evidence must be demonstrated with sufficient information ( *e.g.,* professional guidelines and consistent associations in peer-reviewed published literature). For the selected variants reported as “may be associated with increased risk,” the clinical evidence must be reported in professional guidelines, but peer-reviewed published literature may not be consistent.(D) A statement about the current professional guidelines for testing these specific gene(s) and variant(s) for the specified disease(s). ( *1* ) If professional guidelines are available, provide the recommendations in the professional guideline(s) for the gene, variant, and disease for when genetic testing should or should not be performed, and cautionary information that should be communicated when a particular gene and variant is detected.( *2* ) If professional guidelines are not available, provide a statement that the professional guidelines are not available for these specific gene(s) and variant(s).(iv) The specimen type ( *e.g.,* saliva, whole blood).(v) Assay steps and technology used. (vi) Specification of required ancillary reagents, instrumentation, and equipment. (vii) Specification of the specimen collection, processing, storage, and preparation methods. (viii) Specification of risk mitigation elements and description of all additional procedures, methods, and practices incorporated into the directions for use that mitigate risks associated with testing. (ix) Information pertaining to the probability of test failure ( *e.g.,* percentage of tests that failed quality control) based on data from clinical samples, a description of scenarios in which a test can fail (*e.g.,* low sample volume, low DNA concentration), how users will be notified of a test failure, and the nature of follow-up actions on a failed test to be taken by the user and the manufacturer.(x) When available, information specifying the probability of a false negative and false positive analytical result and any additional considerations by ethnicity. (xi) Specification of the criteria for test result interpretation and reporting, including any distinctions between risk categories ( *i.e.,* increased risk and greatly increased risk; are associated and may be associated).(xii) Information that demonstrates the performance characteristics of the test including: (A) Accuracy of study results for each claimed specimen type. ( *1* ) Accuracy of the test must be evaluated with fresh clinical specimens collected and processed in a manner consistent with the test's instructions for use. If this is impractical, fresh clinical samples may be substituted or supplemented with archived clinical samples. Archived samples must have been collected previously in accordance with the instructions for use, stored appropriately, and randomly selected. In some limited circumstances, use of contrived samples or human cell line samples may also be appropriate and used as an acceptable alternative. The contrived or human cell line samples must mimic clinical specimens as much as is feasible and provide an unbiased evaluation of the test's accuracy.( *2* ) Accuracy must be evaluated by comparison to bidirectional Sanger sequencing or other methods identified as appropriate by FDA. Performance criteria for both the comparator method and the test must be pre-defined and appropriate to the test's intended use. Detailed study protocols must be provided.( *3* ) Information provided must include the number and type of specimens, broken down by clinically relevant variants for each indicated report that were compared to bidirectional sequencing or other methods identified as appropriate by FDA. The accuracy as positive percent agreement (PPA) and negative percent agreement (NPA) must be measured, and accuracy point estimates must be >99 percent (both per reported variant and overall). Uncertainty of the point estimate must be within an acceptable range, as identified by FDA, and must be presented using the 95 percent confidence interval.( *4* ) Sufficient specimens must be tested per genotype and must include all genotypes that will be included in the tests and reports. The number of samples tested in the accuracy study for each variant reported must be based on the variant frequency.( *5* ) Any no calls (*i.e.,* absence of a result) or invalid calls (*e.g.,* failed quality control) in the study must be included in accuracy study results and reported separately. The percent of final 'no calls' or 'invalid calls' must be clinically acceptable. Variants that have a point estimate for PPA or NPA of <99 percent (incorrect test results compared to bidirectional sequencing or other methods identified as appropriate by FDA) must not be incorporated into test claims and reports. Accuracy measures generated from clinical specimens versus contrived samples or cell lines must be presented separately. Results must be summarized and presented in tabular format, by sample and by genotype.( *6* ) Point estimate of PPA for each genotype must be calculated as the number of correct calls for that genotype divided by the number of samples known to contain that genotype. The point estimate of NPA for each genotype must be calculated as the number of correct calls that do not contain that genotype divided by the number of samples known to not contain that genotype. 'No calls' must not be included in these calculations. Point estimates must be calculated along with 95 percent two-sided confidence intervals.(B) Precision and reproducibility data must be provided using multiple instruments and multiple operators, on multiple non-consecutive days, and using multiple reagent lots. The sample panel must include specimens from the claimed sample type ( *e.g.,* saliva) representing all genotypes for each variant (*e.g.,* wild type, heterozygous, and homozygous). Performance criteria must be predefined. A detailed study protocol must be created in advance of the study and then followed. The failed quality control rate must be indicated (*i.e.,* the total number of sample replicates for which a sequence variant cannot be called (no calls) or that fail sequencing quality control criteria divided by the total number of replicates tested). It must be clearly documented whether results were generated from clinical specimens, contrived samples, or cell lines. The study results must state, in a tabular format, the variants tested in the study and the number of replicates for each variant, and what conditions were tested (*e.g.,* number of runs, days, instruments, reagent lots, operators, specimens/type). The study must include all extraction steps from the claimed specimen type or matrix, unless a separate extraction study for the claimed sample type is performed. If the device is to be used at more than one laboratory, different laboratories must be included in the precision study (and reproducibility across sites must be evaluated). Any no calls or invalid calls in the study must be listed as a part of the precision and reproducibility study results.(C) Analytical specificity data: data must be provided evaluating the test performance ( *e.g.,* specimen extraction and variant detection) effect of potential endogenous and exogenous interferents relevant to the specimen type, and assessment of cross-contamination. Alternatively, for each suspected interfering mutation for which data is not provided demonstrating the effect of the interfering variant, the manufacturer must clearly identify the suspected interfering variants in the labeling to user test reports, and indicate that the impact the interfering variants may have on the test's performance has not been studied by providing a statement that reads, “It is possible that the presence of [insert identifying information for the suspected interfering variant] in a sample may interfere with the performance of this test. However, its effect on the performance of this test has not been studied.”(D) Analytical sensitivity data: data must be provided demonstrating the minimum amount of DNA that will enable the test to perform correctly in 95 percent of runs. (E) Device stability data: the manufacturer must establish upper and lower limits of input nucleic acid, sample, and reagent stability that will achieve the test's claimed accuracy and reproducibility. The manufacturer must evaluate stability using wild-type, heterozygous, and homozygous samples. Data supporting such claims must be provided. (F) Specimen type and matrix comparison data: specimen type and matrix comparison data must be generated if more than one specimen type can be tested with this device, including failure rates for the different specimens. (xiii) Clinical Performance Summary. (A) Information to support the clinical performance of each variant in the specific condition which is labeled as “are associated with increased risk” and reported by the test must be provided, as identified in paragraph (b)(4)(iii)(C) of this section. (B) Manufacturers must organize information by the specific variant combination as appropriate ( *e.g.,* wild type, heterozygous, homozygous, compound heterozygous, hemizygous genotypes). For each variant combination, information must be provided in the clinical performance section to support clinical performance for the risk category (*e.g.,* not at risk, increased risk). For each variant combination, a summary of key results must be provided in tabular format or using another method identified as appropriate by FDA to include the appropriate information regarding variant type, data source, definition of the target condition (*e.g.,* disease), clinical criteria for determining whether the target disease is present or absent, description of subjects with the target disease present and target disease absent (exclusion or inclusion criteria), and technical method for genotyping. When available, information on the effect of the variant on risk must be provided as the risk of a disease (lifetime risk or lifetime incidences) for an individual compared with the general population risk.(xiv) User comprehension study: information on a study that assesses comprehension of the test process and results by potential users of the test must be provided, including the following, as appropriate: (A) The test manufacturer must provide a genetic health risk education module to naïve user comprehension study participants prior to their participation in the user comprehension study. The module must define terms that are used in the test reports and explain the significance of genetic risk reports. (B) The test manufacturer must perform pre- and post-test user comprehension studies. The comprehension test questions must directly evaluate the material being presented to the user as described in paragraph (b)(3)(ii) of this section. (C) The manufacturer must provide a justification from a physician and/or genetic counselor that identifies the appropriate general and variant-specific concepts contained within the material being tested in the user comprehension study to ensure that all relevant concepts are incorporated in the study. (D) The user comprehension study must meet the following criteria: ( *1* ) The study participants must comprise a statistically sufficient sample size and demographically diverse population (determined using methods such as quota-based sampling) that is representative of the intended user population. Furthermore, the study participants must comprise a diverse range of age and educational levels and have no prior experience with the test or its manufacturer. These factors must be well-defined in the inclusion and exclusion criteria.( *2* ) All sources of bias (*e.g.,* non-responders) must be predefined and accounted for in the study results with regard to both responders and non-responders.( *3* ) The testing must follow a format where users have limited time to complete the studies (such as an on-site survey format and a one-time visit with a cap on the maximum amount of time that a participant has to complete the tests).( *4* ) Users must be randomly assigned to study arms. Test reports in the user comprehension study given to users must define the target condition being tested and related symptoms, explain the intended use and limitations (including warnings) for the test, explain the relevant ethnicities in regard to the variant tested, explain genetic health risks and relevance to the user's ethnicity, and assess participants' ability to understand the following comprehension concepts: the test's limitations, purpose, appropriate action, test results, and other factors that may have an impact on the test results.( *5* ) Study participants must be untrained, be naïve to the test subject of the study, and be provided the labeling prior to the start of the user comprehension study.( *6* ) The user comprehension study must meet the predefined primary endpoint criteria, including a minimum of a 90 percent or greater overall comprehension rate (*i.e.,* selection of the correct answer) for each comprehension concept. Other acceptance criteria may be acceptable depending on the concept being tested. Meeting or exceeding this overall comprehension rate demonstrates that the materials presented to the user are adequate for over-the-counter use.( *7* ) The analysis of the user comprehension results must include:( *i* ) Results regarding reports that are provided for each gene/variant/ethnicity tested;( *ii* ) Statistical methods used to analyze all data sets; and( *iii* ) Completion rate, non-responder rate, and reasons for nonresponse/data exclusion. A summary table of comprehension rates regarding comprehension concepts (*e.g.,* purpose of test, test results, test limitations, ethnicity relevance for the test results, appropriate actions following receipt of results) for each study report must be included.

Predicate Devices

• 23andMe PGS Risk Report for BRCA1/BRCA2 (Selected Variants) (DEN170046)

Submission Summary (Full Text)

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FDA U.S. FOOD &amp; DRUG ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number

K223597

B Applicant

23andMe, Inc.

C Proprietary and Established Names

23andMe® Personal Genome Service® (PGS®) Cancer Predisposition Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants)

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QAZ | Class II | 21 CFR 866.6090 - Cancer Predisposition Risk Assessment System | Pathology |

## II Submission/Device Overview:

### A Purpose for Submission:

In this Traditional 510(k) submission 23andMe seeks the following:

1. Clearance for an additional 41 BRCA1 and BRCA2 variants to be added to the existing authorized BRCA1/BRCA2 (Selected Variants) report, (DEN170046).
2. Establish a Pre-Determined Change Control Plan (PCCP) for adding additional validated BRCA1 and BRCA2 variants and associated cancer risk information to the report.

### B Measurand:

44 variants (single-nucleotide polymorphism) in the BRCA1 and BRCA2 genes. Refer to the intended use statement for complete list.

### C Type of Test:

Qualitative genetic test for single nucleotide polymorphism detection.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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K223597 - Page 2 of 26

# III Intended Use/Indications for Use:

# A Intended Use(s):

See Indications for Use below.

# B Indication(s) for Use:

The 23andMe Personal Genome Service (PGS) uses qualitative genotyping to detect select clinically relevant variants in genomic DNA isolated from human saliva collected from individuals $\geq 18$ years with the Oragene Dx model OGD500.001 for the purpose of reporting and interpreting genetic health risks, including the 23andMe PGS Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants).

The 23andMe Personal Genome Service (PGS) Risk Report for BRCA1/BRCA2 (Selected Variants) is indicated for the reporting of the following 44 variants in the BRCA1 and BRCA2 genes.

| Gene | Variant(s) |
| --- | --- |
| BRCA1 | c.68_69del, c.213-11T>G, c.427G>T, c.815_824dup, c.1556del, c.1687C>T, c.1960A>T, c.1961del, c.2681_2682del, c.2864C>A, c.3481_3491del, c.3598C>T, c.3627dup, c.3756_3759del, c.3770_3771del, c.4035del, c.4065_4068del, c.4327C>T, c.4357+1G>A, c.4964_4982del, c.4986+6T>G, c.5123C>A, c.5177_5180del, c.5266dup |
| BRCA2 | c.658_659del, c.771_775del, c.1929del, c.2808_2811del, c.2957_2958insG, c.3170_3174del, c.3264dup, c.3545_3546del, c.3847_3848del, c.4471_4474del, c.5542del, c.5576_5579del, c.5682C>G, c.5946del, c.6037A>T, c.6275_6276del, c.7024C>T, c.7480C>T, c.7934del, c.8904del |

The report describes if a person's genetic result is associated with an increased risk of developing breast cancer and ovarian cancer and may be associated with an increased risk for prostate cancer, pancreatic cancer, and potentially other cancers. The variants included in this report do not represent the majority of the BRCA1/BRCA2 variants in people of most ethnicities. The test report does not describe a person's overall risk of developing any type of cancer, and the absence of a variant tested does not rule out the presence of other variants that may be cancer-related. This report is for over-the-counter use by adults over the age of 18, and provides genetic information to inform discussions with a healthcare professional. This test is not a substitute for visits to a healthcare provider for recommended screenings or appropriate follow-up and should not be used to determine any treatments.

# C Special Conditions for Use Statement(s):

1. For over-the-counter (OTC) use.
2. The test does not diagnose cancer or any other health condition and should not be used to make medical decisions. Results should be confirmed in a clinical setting before taking any medical action.

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3. This test is not a substitute for visits to a healthcare provider for recommended screening or appropriate follow-up. It is recommended that users consult with a healthcare provider if there are any questions or concerns about the test results or their current state of health.

4. The 23andMe PGS Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants) detects only three variants and does not detect all genetic variants in these genes associated with increased risk of developing breast, ovarian or prostate cancer. There are more than 1,000 different BRCA1/BRCA2 variants known to be associated with increased risk of developing cancer. The absence of a variant tested does not rule out the presence of other genetic variants that may be disease-related.

5. The test is intended for users ≥ 18 years old.

6. The laboratory may not be able to process a user’s sample. The probability that the laboratory cannot process a sample can be up to 7.6%.

7. A user’s race, ethnicity, age, and sex may affect how the genetic test results are interpreted.

8. It is important for the user to discuss their personal or family history of cancer with a healthcare professional. If the user has a personal or family history of cancer or think they may have symptoms of cancer, the user should consult with their healthcare provider about appropriate testing.

9. Subject to meeting the limitations contained in the special controls under regulation 866.6090.

## D Special Instrument Requirements:

Tecan Evo, Illumina iScan and GenomeStudio system (qualified by the laboratory).

## IV Device/System Characteristics:

### A Device Description:

Customer saliva specimens are self-collected using the Oragene-Dx® Device manufactured by DNA Genotek, Inc. (previously cleared under k141410), which consists of a sealable collection tube containing a stabilizing buffer solution. Specimens are shipped to either one of two laboratories for testing. DNA is isolated from the saliva and tested in a multiplex assay using a customized genotyping beadchip, reagents and instrumentation manufactured by Illumina. The raw data is generated using Illumina GenomeStudio software, and then sent to 23andMe for analysis and interpretation. The raw data received is analyzed using 23andMe’s proprietary Coregen software, where a genotype is determined for each tested SNP. The results for certain of these SNPs are used to generate personalized reports for the customer that provide information about the detected genotype.

Personalized reports are generated for each user that provide results of the testing performed. These reports tell the user which genetic health risk variant(s) have been detected in their sample and provide information about the disease associated with the variant(s). If no variant was detected that information is also provided. The personalized reports are designed to present scientific concepts to users in an easy-to-understand format.

### B Principle of Operation:

The assay uses multiplex microarray technology for the simultaneous detection of variants in human DNA. The BeadChip v5 assay (Illumina Infinium HumanOmniExpress-24 format chip)

K223597 - Page 3 of 26

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consists of silicon wafers etched to form wells loaded with silica beads, on which oligonucleotide capture probes are immobilized. DNA from saliva is fragmented and captured on a bead array by hybridization to immobilized SNP-specific primers, followed by extension with hapten-labeled nucleotides. The primers hybridize adjacent to the SNPs and are extended with a single nucleotide corresponding to the variant allele. The incorporated hapten-modified nucleotides are detected by adding fluorescently labeled antibodies in several steps to amplify the signals. The Tecan Evo and Illumina iScan instruments are used for extraction and processing of the DNA, and the BeadChip for scanning and quantification of the results. The genotype content is separated, analyzed, and then integrated into predefined report templates specific for each condition associated with each genotype. Genotypes are determined using the GenomeStudio and Coregen software packages. For the 23andMe PGS Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants) information on 44 specific variants in the BRCA1/BRCA2 genes are integrated into the report.

## V Substantial Equivalence Information:

### A Predicate Device Name(s):

23andMe PGS Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants)

### B Predicate 510(k) Number(s):

DEN170046

### C Comparison with Predicate(s):

The subject device is similar to the predicate, having similar indications for use, the same intended use, and the same technological characteristics as its predicate device, with the exception of the implementation of a Predetermined Change Control Plan (PCCP) that specifies the protocols and acceptance criteria for making modifications to the reportable BRCA1/BRCA2 variants in a controlled manner, such that the device is as safe and as effective as the predicate.

Specific test methods for clinical and analytical validation are specified in the a to establish substantial equivalence relative to DEN170046, and include sample size determination, analysis methods, and acceptance criteria. The Sponsor will perform testing of the additional BRCA1/2 variants according to the specified protocols, and if the validation data meet the specified acceptance criteria, they may add those variants to the BRCA1/2 report without additional premarket review.

The PCCP is limited to the addition of single nucleotide variants and small insertions and deletions (≤20 bp) in the BRCA1 and BRCA2 genes. The plan describes the specific clinical validation criteria that must be met to demonstrate that the new BRCA1/2 variants are high-risk, highly penetrant BRCA1/2 variants (i.e., those that are demonstrated to be linked to hereditary breast and ovarian cancer (HBOC) syndrome). Specific analytical validation protocols and acceptance criteria are also detailed in the plan to ensure that device maintains the following performance characteristics for each new BRCA1/2 variant:

- Accuracy point estimates of ≥99% positive percent agreement (PPA) and negative percent agreement (NPA), established by comparing results of the 23andMe to bidirectional Sanger sequencing

K223597 - Page 4 of 26

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- $\geq 99\%$ correct genotype calls assigned at each of two laboratory sites
- $\geq 95\%$ of samples yielding the correct genotype call at the minimum DNA input tested. Additionally, software verification and validation activities are detailed in the PCCP, and all must be completed successfully to modify the report to add the new BRCA1/2 variants. The plan specifies change control for genotype calling definitions and labeling updates to ensure the device remains as safe and effective as the predicate device.

Customers who previously opted-in to receive their BRCA1/2 report will receive an email notification informing them that the report has been updated. Customers will have access to their updated report unless they exercise their option to opt-out.

| Device & Predicate Device(s): | K223597 | DEN170046 |
| --- | --- | --- |
| Device Trade Name | 23andMe Personal Genome Service (PGS) Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants) | 23andMe PGS Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants) |
| General Device Characteristics: Similarities | | |
| Intended Use | Same | The 23andMe Personal Genome Service (PGS) uses qualitative genotyping to detect select clinically relevant variants in genomic DNA isolated from human saliva collected from individuals ≥18 years with the Oragene Dx model OGD500.001 for the purpose of reporting and interpreting genetic health risks, including the 23andMe PGS Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants). |
| Specimen Type | Same | Saliva |
| Collection Kit | Same | Oragene·Dx® saliva collection device (OGD-500.001) K141410 |
| BeadChip | Same | Illumina Global Screening Array customized for the PGS. The chip is designed to detect specific single nucleotide polymorphisms (SNPs) as well as other genetic variants; all markers refer to specific positions in the National Center for Biotechnology Information (NCBI) reference human genome. |
| Beadpool | Same | Customized for 23andMe |
| Instruments | Same | Tecan Evo |

K223597 - Page 5 of 26

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| Device & Predicate Device(s): | K223597 | DEN170046 |
| --- | --- | --- |
| | | Illumina iScan |
| Software | Same | Genome Studio
Coregen |
| Differences | | |
| Measurand | 44 variants | 3 variants |
| Reagents | Illumina Infinium HTS
Extra Assay Reagents | Illumina Infinium HTS Assay
Reagents |

VI Standards/Guidance Documents Referenced:

Special controls 21 CFR 866.6090, established in DEN170046

VII Performance Characteristics (if/when applicable):

A Analytical Performance:

1. Precision/Reproducibility:

Precision studies were performed to assess the repeatability and reproducibility of the 23andMe Personal Genome Service (PGS) test for the additional 41 variants to be added to the existing BRCA1/BRCA2 (Selected Variants) report. All 41 variants were included in this study.

This study evaluated intra-assay, inter-lot, inter-instrument, inter-operator, inter-day, and inter-lab precision. Samples were identified from the 23andMe customer database based on their putative genotype and genotyped by the assay in a blinded fashion, with 3 lots of reagents, by multiple operator teams per day, using 3 different serial numbers of each of 2 instruments, over 3 days, at each of 2 laboratory sites. Genotype results were confirmed using bidirectional Sanger sequencing.

There were 2 homozygous common (WT) and 1 heterozygous sample per variant used to evaluate the precision of the assay, with the exception of c.2957_2958insG BRCA2 variant, which included 1 WT and 1 heterozygous sample. Replicates failing quality control acceptance criteria were re-tested per lab procedures. Only sample replicates that passed quality control and produced a genotype for the 23andMe test were included in the calculation for percent agreement.

The precision study yielded greater than 99% correct genotype calls for all samples across two laboratories, on multiple instruments, with multiple operators, on multiple non-consecutive days, and using multiple reagent lots. In addition, the study had greater than 99% reproducibility and greater than 99% repeatability. Results for heterozygous and homozygous common (wild-type) are shown in Table 1. Results with no calls are in bold and shaded in Table 1. FQC refers to Failed Quality Control.

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Table 1. Precision Study Results Stratified by Site and Genotype

| Variant | Genotype | Site | Replicates Pass QC | Correct Calls | Incorrect Calls | No Calls | FQCs | % Correct Calls |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| c.213-11T>G BRCA1 | AA (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AC (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | AA (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AC (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.427G>T BRCA1 | CC (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AC (Heterozygous) | 1 | 81 | 79 | 0 | 2 | 0 | 100% |
| | CC (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AC (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.815_824dup BRCA1 | DD (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | DD (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.1556del BRCA1 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.1687C>T BRCA1 | GG (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AG (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | GG (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AG (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.1960A>T BRCA1 | TT (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AT (Heterozygous) | 1 | 81 | 79 | 0 | 2 | 0 | 100% |

K223597 - Page 7 of 26

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K223597 - Page 8 of 26
| Variant | Genotype | Site | Replicates Pass QC | Correct Calls | Incorrect Calls | No Calls | FQCs | % Correct Calls |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | TT (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| AT (Heterozygous) | 2 | 81 | 80 | 0 | 1 | 0 | 100% |
| c.1961del BRCA1 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 79 | 0 | 2 | 0 | 100% |
| II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.2681_2682del BRCA1 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.2864C>A BRCA1 | GG (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | GT (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| GG (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | GT (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.3481_3491del BRCA1 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 79 | 0 | 2 | 0 | 100% |
| c.3598C>T BRCA1 | GG (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AG (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| GG (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AG (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.3627dup BRCA1 | DD (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |

{8}

K223597 - Page 9 of 26
| Variant | Genotype | Site | Replicates Pass QC | Correct Calls | Incorrect Calls | No Calls | FQCs | % Correct Calls |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | DD (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.3756_3759del BRCA1 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.3770_3771del BRCA1 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.4035del BRCA1 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.4065_4068del BRCA1 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.4327C>T BRCA1 | GG (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AG (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| GG (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AG (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.4357+1G>A BRCA1 | CC (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | CT (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| CC (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |

{9}

K223597 - Page 10 of 26
| Variant | Genotype | Site | Replicates Pass QC | Correct Calls | Incorrect Calls | No Calls | FQCs | % Correct Calls |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | CT (Heterozygous) | 2 | 81 | 80 | 0 | 1 | 0 | 100% |
| c.4964_4982del BRCA1 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.4986+6T>G BRCA1 | AA (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AC (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | AA (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AC (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.5123C>A BRCA1 | GG (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | GT (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | GG (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | GT (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.5177_5180del BRCA1 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.658_659del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.771_775del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |

{10}

K223597 - Page 11 of 26
| Variant | Genotype | Site | Replicates Pass QC | Correct Calls | Incorrect Calls | No Calls | FQCs | % Correct Calls |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| c.1929del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.2808_2811del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.2957_2958insG BRCA2 | DD (Homozygous Common) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | DD (Homozygous Common) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 79 | 0 | 2 | 0 | 100% |
| c.3170_3174del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 80 | 0 | 1 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.3264dup BRCA2 | DD (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | DD (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.3545_3546del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.3847_3848del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |

{11}

K223597 - Page 12 of 26
| Variant | Genotype | Site | Replicates Pass QC | Correct Calls | Incorrect Calls | No Calls | FQCs | % Correct Calls |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.4471_4474del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.5542del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.5576_5579del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 79 | 0 | 2 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 79 | 0 | 2 | 0 | 100% |
| c.5682C>G BRCA2 | CC (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | CG (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | CC (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | CG (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.6037A>T BRCA2 | AA (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AT (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | AA (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | AT (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.6275_6276del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |

{12}

| Variant | Genotype | Site | Replicates Pass QC | Correct Calls | Incorrect Calls | No Calls | FQCs | % Correct Calls |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.7024C>T BRCA2 | CC (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | CT (Heterozygous) | 1 | 81 | 77 | 0 | 4 | 0 | 100% |
| | CC (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | CT (Heterozygous) | 2 | 81 | 78 | 0 | 3 | 0 | 100% |
| c.7480C>T BRCA2 | CC (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | CT (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | CC (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | CT (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.7934del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |
| c.8904del BRCA2 | II (Homozygous Common) | 1 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 1 | 81 | 81 | 0 | 0 | 0 | 100% |
| | II (Homozygous Common) | 2 | 162 | 162 | 0 | 0 | 0 | 100% |
| | DI (Heterozygous) | 2 | 81 | 81 | 0 | 0 | 0 | 100% |

2. Linearity:
Not applicable

3. Analytical Specificity/Interference:
a. Interfering Substances
A series of studies were previously performed to determine whether endogenous substances, exogenous substances, microbial substances, and smoking, affect results of the 23andMe PGS tests. The results of interference studies can be found in the Decision Summary for DEN140044.

K223597 - Page 13 of 26

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# b. Interfering Mutations

The Sponsor identified in the labeling the following rare mutations that may interfere with the performance of the test (Table 2). Interference due to these mutations was not tested. The package insert captures this with a statement indicating that:

"The performance of this test may be affected by the presence of rare mutations, such as those listed below. The effects of these variants on the performance of this test have not been studied."

Table 2. Potentially Interfering Mutations in the BRCA1 and BRCA2 genes

| Gene | Variant Name | Potential Interfering Mutation |
| --- | --- | --- |
| BRCA1 | c.68_69del | rs528170710 |
| | | rs540373654 |
| | | rs80357134 |
| | | rs528902306 |
| | | rs149402012 |
| BRCA1 | c.427G>T | rs371973519 |
| | | rs368415464 |
| | | rs200358748 |
| | | rs542687218 |
| | | rs80356888 |
| BRCA1 | c.815_824dup | rs186274774 |
| | | rs397509328 |
| | | rs8176153 |
| | | rs80357244 |
| | | rs149867679 |
| | | rs201441987 |
| BRCA1 | c.1556del | rs200616937 |
| | | rs56272539 |
| | | rs80357445 |
| BRCA1 | c.1687C>T | rs530914551 |
| | | rs552505690 |
| | | rs80357159 |
| | | rs56012641 |
| BRCA1 | c.1960A>T | rs80356895 |
| | | rs561988641 |
| | | rs28897679 |
| BRCA1 | c.1961del | rs80356895 |
| | | rs561988641 |
| | | rs28897679 |
| BRCA1 | c.2864C>A | rs559190752 |
| | | rs80356851 |

K223597 - Page 14 of 26

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K223597 - Page 15 of 26
| Gene | Variant Name | Potential Interfering Mutation |
| --- | --- | --- |
| BRCA1 | c.3481_3491del | rs56336919 |
| | | rs183119644 |
| | | rs80356918 |
| | | rs80357272 |
| BRCA1 | c.3598C>T | rs537737635 |
| | | rs56214134 |
| | | rs16942 |
| BRCA1 | c.3627dup | rs537737635 |
| | | rs56214134 |
| BRCA1 | c.3756_3759del | rs140588714 |
| | | rs200648498 |
| | | rs483353090 |
| | | rs80357099 |
| | | rs80357191 |
| BRCA1 | c.3770_3771del | rs140588714 |
| | | rs200648498 |
| | | rs483353090 |
| | | rs80357099 |
| | | rs80357191 |
| BRCA1 | c.4035del | rs80357345 |
| | | rs28897689 |
| | | rs80356828 |
| BRCA1 | c.4065_4068del | rs80357345 |
| | | rs28897689 |
| | | rs80356828 |
| BRCA1 | c.4327C>T | rs80358027 |
| | | rs80356840 |
| | | rs1060915 |
| | | rs541512953 |
| BRCA1 | c.4357+1G>A | rs80356840 |
| | | rs1060915 |
| BRCA1 | c.4964_4982del | rs549640262 |
| | | rs1799967 |
| | | rs201810810 |
| | | rs70953661 |
| BRCA1 | c.4986+6T>G | rs549640262 |
| | | rs1799967 |
| | | rs201810810 |
| BRCA1 | c.5123C>A | rs376836050 |
| | | rs397509229 |
| | | rs80356860 |
| BRCA1 | c.5177_5180del | rs8176260 |

{15}

K223597 - Page 16 of 26
| Gene | Variant Name | Potential Interfering Mutation |
| --- | --- | --- |
| | | rs56195342 |
| BRCA1 | c.5266dup | rs371203180 |
| | | rs571834423 |
| BRCA2 | c.658_659del | rs81002855 |
| | | rs568027879 |
| | | rs528919073 |
| BRCA2 | c.771_775del | rs55854959 |
| | | rs549269828 |
| | | rs567889781 |
| BRCA2 | c.1929del | rs11571652 |
| | | rs28897711 |
| | | rs527579384 |
| BRCA2 | c.2808_2811del | rs2227943 |
| | | rs28897716 |
| | | rs149753706 |
| | | rs80358535 |
| BRCA2 | c.2957_2958insG | rs45525041 |
| | | rs539613324 |
| | | rs144862123 |
| | | rs11571656 |
| | | rs80358541 |
| | | rs2227944 |
| | | rs1799944 |
| BRCA2 | c.3170_3174del | rs564316199 |
| | | rs145605603 |
| BRCA2 | c.3264dup | rs145605603 |
| | | rs543748012 |
| | | rs80358575 |
| BRCA2 | c.3545_3546del | rs80358600 |
| | | rs1799952 |
| BRCA2 | c.3847_3848del | rs543304 |
| | | rs41293485 |
| BRCA2 | c.5542del | rs138489917 |
| | | rs372951842 |
| | | rs573514896 |
| BRCA2 | c.5576_5579del | rs372951842 |
| | | rs573514896 |
| | | rs80358782 |
| BRCA2 | c.5682C>G | rs55996097 |
| | | rs11571657 |
| | | rs146351301 |
| | | rs4987048 |

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| Gene | Variant Name | Potential Interfering Mutation |
| --- | --- | --- |
| | | rs149474191 |
| | | rs55875643 |
| BRCA2 | c.5946del | rs556893517 |
| | | rs148618542 |
| | | rs80358833 |
| | | rs554663691 |
| BRCA2 | c.6037A>T | rs554663691 |
| | | rs572976024 |
| | | rs540799830 |
| | | rs147961615 |
| BRCA2 | c.6275_6276del | rs541826447 |
| | | rs397507838 |
| | | rs55794205 |
| | | rs35029074 |
| | | rs79456940 |
| BRCA2 | c.7024C>T | rs186220967 |
| | | rs45574331 |
| | | rs80358932 |
| | | rs200078639 |
| BRCA2 | c.7480C>T | rs80358965 |
| | | rs11571707 |
| | | rs55716624 |
| | | rs56070345 |
| BRCA2 | c.7934del | rs529779203 |
| BRCA2 | c.8904del | rs59004709 |

4. Assay Reportable Range:
Not applicable

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
The traceability, stability and expected values for the device were previously reviewed in DEN140044. The assay requires two types of controls: the sample processing control and the reproducibility control. The information provided demonstrates that the sample processing control is stable for up to three months and the reproducibility control is stable for up to 12 months. See DEN140044 for detailed information.

6. Detection Limit:
A minimum DNA input study was performed to determine the lowest concentration of DNA that is necessary for successful assignment of correct genotypes for the additional 41 variants

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to be added to the existing BRCA1/BRCA2 (Selected Variants) report. All 41 variants were included in this study.

Samples for this study were selected from the 23andMe customer database based on their putative genotype and included homozygous common (1 or more sample(s) per variant) and heterozygous common (1 sample per variant) genotypes for each variant. The sample input for the assay is $15\mathrm{ng} / \mu \mathrm{L}$ . Therefore, samples were diluted to 3 different concentrations (5, 15, and $50\mathrm{ng} / \mu \mathrm{L}$ ) and genotyped by the assay in a blinded fashion in triplicates using 3 lots of reagents. Genotype results were confirmed using bidirectional Sanger sequencing. The minimum DNA requirement was defined as the lowest concentration at which at least $95\%$ of samples yield the correct call. This minimum DNA input study yielded $100\%$ correct genotype calls for all samples and reagent lots tested at all sample DNA concentrations. Therefore, the study passed the acceptance criteria of $95\%$ correct calls at the lowest concentration tested ( $5\mathrm{ng} / \mu \mathrm{L}$ ). The performance requirement, specified by contract laboratory SOPs, is conservatively set at a minimum of $15\mathrm{ng} / \mu \mathrm{L}$ and a maximum of $50\mathrm{ng} / \mu \mathrm{L}$ .

# 7. Assay Cut-Off:

Not applicable

# B Comparison Studies:

# 1. Comparison with Sanger bidirectional Sequencing:

Accuracy was evaluated through calculation of agreement of the genetic variant determinations between the 23andMe PGS test results and Sanger bidirectional sequencing (comparator) results. All additional 41 variants to be added to the existing BRCA1/BRCA2 (Selected Variants) report were included in the study.

Saliva samples were randomly selected from the 23andMe customer biobank based on predetermined genotypes and the minimum volume required for testing. All chosen samples were then genotyped using Sanger bidirectional sequencing. All Sanger bidirectional sequencing was performed at an independent laboratory site. Genotyping results were compared between the PGS test and bidirectional sequencing to calculate percent agreements with the sequencing results used as the reference. The accuracy data generated for each test report met the Manufacturer's pre-defined acceptance criteria: a minimum of $99\%$ positive percent agreement (PPA) and negative percent agreement (NPA) for each genotype. All results were correct except for BRCA2c.3847_3848del Heterozygous which had one incorrect false negative call. There were 4 no calls. The widest $95\%$ confidence interval was $47.3\%$ to $100\%$ for the heterozygous c.2864C&gt;A BRCA1 genotype. The data is shown in Table 3.

Table 3. Percent Agreement for BRCA1/BRCA2 Variants by Genotypes

| Genotype by Sanger | PGS Genotype Call | | | | % PPA | % NPA | 95% CI** |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Correct* | Incorrect* | No Call | FQC | | | |
| BRCA1 c.213-11T>G Homozygous Common | 35 | 0 | 0 | 0 | N/A | 100% | 91.8% - 100% |

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| Genotype by Sanger | PGS Genotype Call | | | | % PPA | % NPA | 95% CI** |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Correct* | Incorrect* | No Call | FQC | | | |
| BRCA1 c.213-11T>G
Heterozygous | 15 | 0 | 0 | 0 | 100% | N/A | 81.9% - 100% |
| BRCA1 c.427G>T
Homozygous Common | 21 | 0 | 0 | 0 | N/A | 100% | 86.7% - 100% |
| BRCA1 c.427G>T
Heterozygous | 27 | 0 | 0 | 0 | 100% | N/A | 89.5% - 100% |
| BRCA1c.815_824dup
Homozygous Common | 22 | 0 | 0 | 0 | N/A | 100% | 87.3% - 100% |
| BRCA1c.815_824dup
Heterozygous | 26 | 0 | 0 | 0 | 100% | N/A | 89.1% - 100% |
| BRCA1c.1556del
Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA1c.1556del
Heterozygous | 26 | 0 | 0 | 0 | 100% | N/A | 89.1% - 100% |
| BRCA1 c.1687C>T
Homozygous Common | 23 | 0 | 0 | 0 | N/A | 100% | 87.8% - 100% |
| BRCA1 c.1687C>T
Heterozygous | 22 | 0 | 0 | 0 | 100% | N/A | 87.3% - 100% |
| BRCA1 c.1960A>T
Homozygous Common | 29 | 0 | 0 | 0 | N/A | 100% | 90.2% - 100% |
| BRCA1 c.1960A>T
Heterozygous | 25 | 0 | 1 | 0 | 100% | N/A | 88.7% - 100% |
| BRCA1c.1961del
Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA1c.1961del
Heterozygous | 13 | 0 | 0 | 0 | 100% | N/A | 79.4% - 100% |
| BRCA1c.2681_2682del
Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA1c.2681_2682del
Heterozygous | 22 | 0 | 0 | 0 | 100% | N/A | 87.3% - 100% |
| BRCA1 c.2864C>A
Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA1 c.2864C>A
Heterozygous | 4 | 0 | 0 | 0 | 100% | N/A | 47.3% - 100% |
| BRCA1c.3481_3491del
Homozygous Common | 26 | 0 | 0 | 0 | N/A | 100% | 89.1% - 100% |
| BRCA1c.3481_3491del
Heterozygous | 29 | 0 | 0 | 0 | 100% | N/A | 90.2% - 100% |
| BRCA1 c.3598C>T
Homozygous Common | 21 | 0 | 0 | 0 | N/A | 100% | 86.7% - 100% |
| BRCA1 c.3598C>T
Heterozygous | 24 | 0 | 0 | 0 | 100% | N/A | 88.3% - 100% |
| BRCA1c.3627dup
Homozygous Common | 22 | 0 | 0 | 0 | N/A | 100% | 87.3% - 100% |

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| Genotype by Sanger | PGS Genotype Call | | | | % PPA | % NPA | 95% CI** |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Correct* | Incorrect* | No Call | FQC | | | |
| BRCA1c.3627dup Heterozygous | 12 | 0 | 0 | 0 | 100% | N/A | 77.9% - 100% |
| BRCA1c.3756_3759del Homozygous Common | 22 | 0 | 0 | 0 | N/A | 100% | 87.3% - 100% |
| BRCA1c.3756_3759del Heterozygous | 23 | 0 | 0 | 0 | 100% | N/A | 87.8% - 100% |
| BRCA1c.3770_3771del Homozygous Common | 22 | 0 | 0 | 0 | N/A | 100% | 87.3% - 100% |
| BRCA1c.3770_3771del Heterozygous | 27 | 0 | 0 | 0 | 100% | N/A | 89.5% - 100% |
| BRCA1c.4035del Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA1c.4035del Heterozygous | 23 | 0 | 0 | 0 | 100% | N/A | 87.8% - 100% |
| BRCA1c.4065_4068del Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA1c.4065_4068del Heterozygous | 21 | 0 | 0 | 0 | 100% | N/A | 86.7% - 100% |
| BRCA1 c.4327C>T Homozygous Common | 26 | 0 | 0 | 0 | N/A | 100% | 89.1% - 100% |
| BRCA1 c.4327C>T Heterozygous | 24 | 0 | 0 | 0 | 100% | N/A | 88.3% - 100% |
| BRCA1 c.4357+1G>A Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA1 c.4357+1G>A Heterozygous | 8 | 0 | 0 | 0 | 100% | N/A | 68.8% - 100% |
| BRCA1c.4964_4982del Homozygous Common | 30 | 0 | 0 | 0 | N/A | 100% | 90.5% - 100% |
| BRCA1c.4964_4982del Heterozygous | 20 | 0 | 0 | 0 | 100% | N/A | 86.1% - 100% |
| BRCA1 c.4986+6T>G Homozygous Common | 27 | 0 | 0 | 0 | N/A | 100% | 89.5% - 100% |
| BRCA1 c.4986+6T>G Heterozygous | 14 | 0 | 0 | 0 | 100% | N/A | 80.7% - 100% |
| BRCA1 c.5123C>A Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA1 c.5123C>A Heterozygous | 29 | 0 | 0 | 0 | 100% | N/A | 90.2% - 100% |
| BRCA1c.5177_5180del Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA1c.5177_5180del Heterozygous | 26 | 0 | 0 | 0 | 100% | N/A | 89.1% - 100% |
| BRCA2c.658_659del Homozygous Common | 26 | 0 | 0 | 0 | N/A | 100% | 89.1% - 100% |

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| Genotype by Sanger | PGS Genotype Call | | | | % PPA | % NPA | 95% CI** |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Correct* | Incorrect* | No Call | FQC | | | |
| BRCA2c.658_659del Heterozygous | 20 | 0 | 0 | 0 | 100% | N/A | 86.1% - 100% |
| BRCA2c.771_775del Homozygous Common | 23 | 0 | 0 | 0 | N/A | 100% | 87.8% - 100% |
| BRCA2c.771_775del Heterozygous | 25 | 0 | 0 | 0 | 100% | N/A | 88.7% - 100% |
| BRCA2c.1929del Homozygous Common | 26 | 0 | 0 | 0 | N/A | 100% | 89.1% - 100% |
| BRCA2c.1929del Heterozygous | 25 | 0 | 0 | 0 | 100% | N/A | 88.7% - 100% |
| BRCA2c.2808_2811del Homozygous Common | 48 | 0 | 0 | 0 | N/A | 100% | 93.9% - 100% |
| BRCA2c.2808_2811del Heterozygous | 31 | 0 | 0 | 0 | 100% | N/A | 90.8% - 100% |
| BRCA2c.2957_2958insG Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA2c.2957_2958insG Heterozygous | 6 | 0 | 0 | 0 | 100% | N/A | 60.7% - 100% |
| BRCA2c.3170_3174del Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA2c.3170_3174del Heterozygous | 26 | 0 | 0 | 0 | 100% | N/A | 89.1% - 100% |
| BRCA2c.3264dup Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA2c.3264dup Heterozygous | 27 | 0 | 0 | 0 | 100% | N/A | 89.5% - 100% |
| BRCA2c.3545_3546del Homozygous Common | 27 | 0 | 0 | 0 | N/A | 100% | 89.5% - 100% |
| BRCA2c.3545_3546del Heterozygous | 31 | 0 | 0 | 0 | 100% | N/A | 90.8% - 100% |
| BRCA2c.3847_3848del Homozygous Common | 102 | 0 | 0 | 0 | N/A | 100% | 97.1% - 100% |
| BRCA2c.3847_3848del Heterozygous | 108 | 1 | 0 | 0 | 99.03% | N/A | 95.56% - 99.96% |
| BRCA2c.4471_4474del Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA2c.4471_4474del Heterozygous | 5 | 0 | 0 | 0 | 100% | N/A | 54.9% - 100% |
| BRCA2c.5542del Homozygous Common | 25 | 0 | 0 | 0 | N/A | 100% | 88.7% - 100% |
| BRCA2c.5542del Heterozygous | 6 | 0 | 0 | 0 | 100% | N/A | 60.7% - 100% |
| BRCA2c.5576_5579del Homozygous Common | 22 | 0 | 0 | 0 | N/A | 100% | 87.3% - 100% |

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| Genotype by Sanger | PGS Genotype Call | | | | % PPA | % NPA | 95% CI** |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Correct* | Incorrect* | No Call | FQC | | | |
| BRCA2c.5576_5579del Heterozygous | 26 | 0 | 0 | 0 | 100% | N/A | 89.1% - 100% |
| BRCA2 c.5682C>G Homozygous Common | 26 | 0 | 0 | 0 | N/A | 100% | 89.1% - 100% |
| BRCA2 c.5682C>G Heterozygous | 27 | 0 | 0 | 0 | 100% | N/A | 89.5% - 100% |
| BRCA2 c.6037A>T Homozygous Common | 22 | 0 | 0 | 0 | N/A | 100% | 87.3% - 100% |
| BRCA2 c.6037A>T Heterozygous | 17 | 0 | 2 | 0 | 100% | N/A | 83.8% - 100% |
| BRCA2c.6275_6276del Homozygous Common | 25 | 0 | 0 | 0 | N/A | 100% | 88.7% - 100% |
| BRCA2c.6275_6276del Heterozygous | 25 | 0 | 1 | 0 | 100% | N/A | 88.7% - 100% |
| BRCA2 c.7024C>T Homozygous Common | 29 | 0 | 0 | 0 | N/A | 100% | 90.2% - 100% |
| BRCA2 c.7024C>T Heterozygous | 9 | 0 | 0 | 0 | 100% | N/A | 71.7% - 100% |
| BRCA2 c.7480C>T Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA2 c.7480C>T Heterozygous | 25 | 0 | 0 | 0 | 100% | N/A | 88.7% - 100% |
| BRCA2c.7934del Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA2c.7934del Heterozygous | 15 | 0 | 0 | 0 | 100% | N/A | 81.9% - 100% |
| BRCA2c.8904del Homozygous Common | 24 | 0 | 0 | 0 | N/A | 100% | 88.3% - 100% |
| BRCA2c.8904del Heterozygous | 26 | 0 | 0 | 0 | 100% | N/A | 89.1% - 100% |

*Relative to Sanger Sequencing
**calculated using the mid-p method

2. Matrix Comparison:

Not applicable. This test is for use with human saliva samples only.

C Clinical Studies:

1. Disease Description and Clinical Summary

The 23andMe PGS® Genetic Health Risk Test for BRCA1/BRCA2 (Selected Variants) is indicated for the detection of 44 variants in the BRCA1 and BRCA2 genes. These variants are associated with an increased risk for female breast and ovarian cancer (including early-onset breast cancer) and male breast cancer and may be associated with an increased risk for prostate cancer, pancreatic cancer, and potentially other cancers (NCCN 2023). The clinical validity of

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the BRCA1/2 variants is supported by clinical guidelines, public and proprietary databases, published data, and frequency data from the 23andMe customer database.

Pathogenic BRCA1 and BRCA2 variants are highly penetrant: for example, females with a variant have a $45 - 85\%$ chance of developing breast cancer and up to a $46\%$ chance of developing ovarian cancer by age 70 (ACOG 2017, Obstet Gynecol. 2017 Sept, 130 (3): e110-e126.PMID: 28832484).

The overall prevalence of pathogenic variants in BRCA1 and BRCA2 is estimated to be between 1 in 300 and 1 in 800 in the general population (ACOG 2017, Obstet Gynecol. 2017 Sept, 130 (3): e110-e126.PMID: 28832484), and thousands of unique variants have been identified (BRCA Exchange; ACOG 2017, Obstet Gynecol. 2017 Sept, 130 (3): e110-e126.PMID: 28832484; Petrucelli et al, Genet Med 2010,12(5):245-59. PMID: 20216074).

The variants included in the modified BRCA1/BRCA2 (Selected Variants) report account for more than $90\%$ of cancer-related BRCA1 and BRCA2 variants among people of Ashkenazi Jewish descent; about $30 - 40\%$ among African Americans, people of European descent, and people of Hispanic or Latino descent; about $5 - 25\%$ among people of East Asian descent; and up to $35\%$ among people of South Asian descent (Bhaskaran et al, J. Med Genet 2021 Nov, 58 (11):752-759. PMID 32963034).

The table below summarizes the population risk for certain cancers provided in the 23andMe PGS BRCA1/BRCA2 (Selected variants) test reports. The report provides risk estimates for several cancers in individuals with any pathogenic variant in the BRCA1 or BRCA2 gene rather than estimates specific to the variant detected.

Table 4. Numerical and qualitative risk estimates provided in the 23andMe PGS BRCA1/BRCA2 (Selected Variants) test reports

| Cancer Type | General population | BRCA1 variant | BRCA2 variant |
| --- | --- | --- | --- |
| Breast (female) | 12.9% | 45-85% | 45-85% |
| Ovarian | 1.1% | 39-46% | 10-27% |
| Breast (male) | 0.1% | 1-2% | 7-8% |
| Prostate | 12.6% | May have an increased risk* | Increased risk |
| Pancreatic | 1.7% | May have an increased risk | Increased risk |
| Melanoma | 2.1% | Research ongoing | Research ongoing |

* For males with a BRCA1 variant, some studies did not observe an increased risk for prostate cancer

Risk estimates in the table above are based on the following references:

- Committee on Practice Bulletins-Gynecology, Committee on Genetics, Society of Gynecologic Oncology. (2017). "Practice Bulletin No 182: Hereditary Breast and Ovarian Cancer Syndrome." Obstet Gynecol. 130(3):e110-e126.
- Evans DG et al. (2010). "Risk of breast cancer in male BRCA2 carriers." J Med Genet. 47(10): 10-1.
- Surveillance Research Program, National Cancer Institute. "SEER*Explorer: An interactive website for SEER cancer statistics." 2017-2019. Retrieved Dec 6, 2022, from https://seer.cancer.gov/statistics-network/explorer/

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- Tai YC et al. (2007). "Breast cancer risk among male BRCA1 and BRCA2 mutation carriers." J Natl Cancer Inst. 99(23):1811-4.

2. Clinical Sensitivity:
Not applicable

3. Clinical Specificity:
Not applicable

4. Other Clinical Supportive Data:

i. User Comprehension Study

Specific user comprehension studies were not performed to specifically assess the comprehension of the Genetic Health Risk report for BRCA1/BRCA2 (Selected Variants). To fulfill special control 21CFR 866.6090 (b) (4) (xiv), 23andMe provided real world data that included customer service records, adverse event reports, Corrective and Preventive Actions (CAPA) documentation, user feedback, and ethnicity information, collected since the marketing authorization date of each of the cancer predisposition risk reports (DEN170046, K182784, K211499) up to July 13, 2023. The study population consisted of users who had opted in to receive their Cancer Predisposition Risk reports. Less than 0.1% (one-tenth of one percent) of the total number of customers who viewed each Cancer Predisposition Risk report had customer contacts or complaints related to the comprehension concepts for each type of report. There were no adverse events (zero) related to the Cancer Predisposition Risk reports, and there had been only 2 (two) complaints related to user comprehension for which corrective actions resolved. Overall, it was determined that the data was not conclusive of the user comprehension because it excludes customers who may have contacted a health care professional to resolve their questions, or customers who don't realize they misunderstood the report. However, it was determined that the test has appropriate mitigations in place to address potential false interpretation of the results. These mitigations include warnings in the customer report that indicate that the results should be shared with a healthcare professional, the results should be confirmed by an independent genetic test prescribed by your own healthcare provider before taking any medical action, and that the test only includes 44 out of more than 4000 variants in the BRCA1 and BRCA2 genes that are known to increase cancer risk and customer could still have a variant not included in this test. These warnings and limitations are present in several areas of the report (report overview page, scientific details page, FAQ section, and package insert) and provide users with information to adequately understand the purpose, limitations and meaning of the results of the test.

ii. Frequently Asked Questions Material

The Manufacturer has developed a Frequently Asked Questions (FAQ) section for the BRCA1/BRCA2 (Selected Variants) Genetic Health Risk (GHR) report, which is included in the test report and accessible to the user on the Manufacturer's public website. The FAQs are specific to the variants and disease risk associations being reported, where applicable. The FAQ section was created to provide users with information to adequately understand the purpose, limitations and meaning of the results of the test. The FAQ section was developed using methodology consistent with the

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Manufacturer's labeling design, identification of primary communication messages, and label comprehension. The concepts covered in the FAQ section include: the test results, purpose of the test, limitations of the test, relevance of race and ethnicity on test results, meaning of the result, other risk factors that contribute to disease, appropriate follow-up procedures, how the results of the test may affect the user's family and children, and links to resources that provide additional information. Additionally, the FAQ section provides definitions for terminology found in Genetic Health Risk Reports that is used to describe risks associated with detected variants. The specific questions and answers provided are customized based on the customer's result.

iii. User Opt-In Page

Prior to receiving the test results, a pre-purchase page informs users that there is a choice of whether or not to receive the BRCA1/BRCA2 (Selected Variants) test report. Users have an opportunity to opt into receiving these results after reviewing important information included in an opt-in page. The opt-in page is provided for the BRCA1/BRCA2 (Selected Variants) GHR report users due to the nature of the diseases and associated risks for this report, the availability of risk-reducing surgery or medication available for individuals who carry BRCA1 or BRCA2 variants, and the fact that this test is not designed to inform clinical decision-making. Users will be directed to a page entitled, "Choose your health reports" which provides the option to exclude this report from the user's account. The report selection page includes important information to allow the users to make an informed decision. Results of the BRCA1/BRCA2 (Selected Variants) report are locked by default. Customers must elect to opt-in to receive cancer predisposition risk reports and are able to update their report opt-in selections at any time within their account settings. Existing customers who have already opted in to the BRCA1/BRCA2 (Selected Variants) report will not be prompted to opt in again after the report labeling has been modified. New customers, or customers who have not previously opted in, will be required to opt in to the BRCA1/BRCA2 (Selected Variants) report prior to viewing their results, as authorized in DEN170046. All customers must complete the revised education module in order to view the report. Until this is completed, report results will not be viewable within a customer's account.

D. Clinical Cut-Off:

Not applicable

E. Expected Values/Reference Range:

The variants included in the report cover more than 90% of pathogenic BRCA variants in individuals of Ashkenazi Jewish descent (Janavicius et al, EPMA J, (2010), (3):397-412. PMID: 23199084; Rosenthal et al, Breast Cancer Res Treat. (2015), 149 (1): 223-7. PMID: 25476495); about 30-40% in people of African American, European, and Hispanic/Latino descent (Rebbeck et al, Hum Mutat. (2018), 39 (5): 593-620. PMID 29446198); about 5-25% among people of East Asian descent; and up to 35% among people of South Asian descent (Bhaskaran et al, J Med Genet. (2021), Nov; 58 (11): 752-759. PMID 32963034).

The user test reports include allele frequencies from 23andMe customers in the Scientific Details Page. These allele frequencies are from the 23andMe database and may not be representative for the actual allele frequencies.

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VIII Proposed Labeling:

The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:

The subject device, 23andMe Personal Genome Service (PGS) Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants), has similar indications for use, the same intended use, and the same principles of operation as its predicate device, DEN170046. The technological characteristics are identical with the exception of the implementation of a PCCP, and this difference does not raise new questions of safety and effectiveness. Thus, the subject device is substantially equivalent.

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.