23andMe Personal Genome Service (PGS) Genetic Health Risk Test for Hereditary Thrombophilia

DEN160026 · 23AndMe, Inc. · PTA · Apr 6, 2017 · Immunology

Device Facts

Record IDDEN160026
Device Name23andMe Personal Genome Service (PGS) Genetic Health Risk Test for Hereditary Thrombophilia
Applicant23AndMe, Inc.
Product CodePTA · Immunology
Decision DateApr 6, 2017
DecisionDENG
Submission TypeDirect
Regulation21 CFR 866.5950
Device ClassClass 2
AttributesSoftware as a Medical Device

Intended Use

The 23andMe Personal Genome Service (PGS) Test uses qualitative genotyping to detect the following clinically relevant variants in genomic DNA isolated from human saliva collected from individuals ≥18 years with the Oragene Dx model OGD-500.001 for the purpose of reporting and interpreting Genetic Health Risks (GHR): The 23andMe PGS Genetic Health Risk Report for Hereditary Thrombophilia is indicated for reporting of the Factor V Leiden variant in the F5 gene, and the Prothrombin G20210A variant in the F2 gene. This report describes if a person has variants associated with a higher risk of developing harmful blood clots, but it does not describe a person's overall risk of developing harmful blood clots. This test is most relevant for people of European descent. The 23andMe PGS Genetic Health Risk Report for Alpha-1 Antitrypsin Deficiency is indicated for reporting of the PI*Z and PI*S variants in the SERPINA1 gene. This report describes if a person has variants associated with AAT deficiency and a higher risk for lung or liver disease, but it does not describe a person's overall risk of developing lung or liver disease. This test is most relevant for people of European descent. The 23andMe PGS Genetic Health Risk Report for Late-onset Alzheimer’s Disease is indicated for reporting of the ε4 variant in the APOE gene. The report describes if a person's genetic result is associated with an increased risk of developing Late-onset Alzheimer’s Disease, but it does not describe a person's overall risk of developing Alzheimer’s Disease. The ε4 variant included in this report is found and has been studied in many ethnicities. Detailed risk estimates have been studied the most in people of European descent. The 23andMe PGS Genetic Health Risk Report for Parkinson’s Disease is indicated for reporting of the G2019S variant in the LRRK2 gene and the N370S variant in the GBA gene. The report describes if a person's genetic result is associated with an increased risk of developing Parkinson’s disease, but it does not describe a person's overall risk of developing Parkinson’s disease. The test is most relevant for people of European, Ashkenazi Jewish, and North African Berber descent. The 23andMe PGS Genetic Health Risk Report for Gaucher Disease Type 1 is indicated for reporting of the N370S, 84GG, and V394L variants in the GBA gene. This report describes if a person has variants associated with an increased risk for developing symptoms of Gaucher Disease Type 1, but it does not describe a person's overall risk of developing Gaucher Disease Type 1. This test is most relevant for people of Ashkenazi Jewish descent. The 23andMe PGS Genetic Health Risk Report for Factor XI Deficiency is indicated for reporting of the variants F283L, E117X, IVS14+1G>A in the F11 gene. This report describes if a person has a variant associated with Factor XI deficiency and the potential for a higher risk of excessive bleeding following trauma or surgery, but it does not describe a person's overall risk for excessive bleeding. This test is most relevant for people of Ashkenazi Jewish descent. The 23andMe PGS Genetic Health Risk Report for Celiac Disease is indicated for reporting of a variant associated with the HLA-DQ2.5 haplotype. The report describes if a person has a haplotype associated with an increased risk of developing celiac disease, but it does not describe a person's overall risk for developing celiac disease. This report is most relevant for people of European descent. The 23andMe PGS Genetic Health Risk Report for Glucose-6-Phosphate-Dehydrogenase Deficiency is indicated for reporting of the Val68Met variant in the G6PD gene. This report describes if a person has a variant associated with G6PD deficiency and a higher risk for episodes of anemia, but it does not describe a person's overall risk of developing anemia. This test is most relevant for people of African descent. The 23andMe PGS Genetic Health Risk Report for Hereditary Hemochromatosis is indicated for reporting of the C282Y and H63D variants in the HFE gene. This report describes if a person has variants associated with hereditary hemochromatosis and a higher risk for iron overload, but it does not describe a person’s overall risk of developing iron overload. This report is most relevant for people of European descent. The 23andMe PGS Genetic Health Risk Report for Early-Onset Primary Dystonia (DYT1/TOR1A-Related) is indicated for reporting of the deltaE302/303 variant in the DYT1 gene. This report describes if a person has variants associated with a higher risk for early-onset primary dystonia, but it does not describe a person’s overall risk of developing dystonia. This report is most relevant for people of Ashkenazi Jewish descent.

Device Story

Direct-to-consumer, over-the-counter genetic testing service. Input: human saliva sample self-collected in Oragene-Dx tube. Processing: DNA isolation at CLIA-certified labs; multiplex assay on Illumina genotyping chip (>500,000 variants). Analysis: Illumina GenomeStudio software generates raw data; 23andMe proprietary Coregen software determines genotypes. Output: personalized reports detailing detected variants and associated disease risks. Used by consumers to inform lifestyle choices and discussions with healthcare providers. Benefits: provides individuals access to genetic risk information for specific conditions, potentially enabling early informed lifestyle adjustments or clinical surveillance. Does not diagnose conditions or provide overall risk assessment.

Clinical Evidence

Clinical performance supported by published meta-analyses and peer-reviewed literature for each variant. Analytical performance (precision/reproducibility, LoD, accuracy) validated against Sanger bidirectional sequencing. Accuracy studies showed high percent agreement (PA) for all variants. User comprehension study (n=694) demonstrated >90% overall comprehension of test reports.

Technological Characteristics

Qualitative in vitro molecular diagnostic system; detects specific SNPs/variants in genomic DNA; utilizes saliva specimen collection (Oragene Dx OGD-500.001); requires standardized nomenclature (HUGO, dbSNP); performance validated via accuracy, precision, and reproducibility studies; includes web-based reporting and educational interface.

Indications for Use

Indicated for qualitative genotyping of specific clinically relevant variants in genomic DNA from human saliva (collected via Oragene Dx OGD-500.001) in individuals ≥18 years to report/interpret genetic health risks. Not for diagnosis; not a substitute for healthcare provider visits.

Regulatory Classification

Identification

A genetic health risk assessment system is a qualitative in vitro molecular diagnostic system used for detecting variants in genomic deoxyribonucleic acid (DNA) isolated from human specimens that will provide information to users about their genetic risk of developing a disease to inform lifestyle choices and/or conversations with a health care professional. This assessment system is for over-the-counter use. This device does not determine the person's overall risk of developing a disease.

Special Controls

*Classification.* Class II (special controls). The genetic health risk assessment system device, when it has previously received a first-time FDA marketing authorization (*e.g.,* 510(k) clearance) for the genetic health risk assessment system (a “one-time FDA reviewed genetic health risk assessment system”), is exempt from the premarket notification procedures in part 807, subpart E, of this chapter subject to the limitations in § 866.9. The device must comply with the following special controls:(1) The 21 CFR 809.10 compliant labeling and any prepurchase page and test report generated, unless otherwise specified, must include: (i) A section addressed to users with the following information: (A) The limiting statement explaining that this test provides genetic risk information based on assessment of specific genetic variants but does not report on a user's entire genetic profile. This test [does not/may not, as appropriate] detect all genetic variants related to a given disease, and the absence of a variant tested does not rule out the presence of other genetic variants that may be related to the disease. (B) The 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. (C) The limiting statement explaining that other factors such as environmental and lifestyle risk factors may affect the risk of developing a given disease. (D) The 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 health care professional prior to collection of a sample for testing. This test is not a substitute for visits to a doctor or other health care professional. Users should consult with their doctor or other health care professional if they have any questions or concerns about the results of their test or their current state of health. (E) Information about how to obtain access to a genetic counselor, board-certified clinical molecular geneticist, or equivalent health care professional about the results of a user's test. (F) The limiting statement explaining that this test is not intended to diagnose a disease, 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. (G) 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. (ii) A section in your 21 CFR 809.10 labeling and any test report generated that is for health care professionals who may receive the test results from their patients with the following information: (A) The limiting statement explaining that this test is not intended to diagnose a disease, determine medical treatment, or tell the user anything about their current state of health. (B) The limiting statement explaining that this test is intended to provide users with their genetic information to inform lifestyle decisions and conversations with their doctor or other health care professional. (C) The limiting statement explaining that any diagnostic or treatment decisions should be based on testing and/or other information that you determine to be appropriate for your patient. (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 Web site where the manufacturer shall 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 Web site 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 Web site or testing using the device can be ordered from the Web site, 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 would also have to be posted on the manufacturer's Web site. 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) 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. If there are different categories of risk, the manufacturer must provide literature references 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, but is not limited to, 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* )*Prepurchase page:* This page must contain information that informs the user about what information the test will provide. This includes, but is not limited to, variant information, the condition or disease 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) 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 section must be provided. This opt-in page must be provided for each disease 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* ) Professional guidelines that recommend when genetic testing for the associated target condition is or is not recommended; and( *iv* ) A recommendation to speak with a health care professional, genetic counselor, or equivalent professional before getting the results of the test.( *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 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 followup procedures, how the results of the test may affect the user's family, including children, and links to resources that provide additional information.(iii) A technical information section containing the following information: (A) Gene(s) and variant(s) the test detects using standardized nomenclature, Human Genome Organization nomenclature and coordinates as well as Single Nucleotide Polymorphism Database (dbSNP) reference SNP numbers (rs#). (B) Scientifically established disease-risk association of each variant detected and reported by the test. This risk association information must include: ( *1* ) Genotype-phenotype information for the reported variants.( *2* ) Table of expected frequency and risks of developing the disease in relevant ethnic populations and the general population.( *3* ) A statement about the current professional guidelines for testing these specific gene(s) and variant(s).( *i* ) If professional guidelines are available, provide the recommendations in the professional guideline 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.( *ii* ) If professional guidelines are not available, provide a statement that the professional guidelines are not available for these specific gene(s) and variant(s).(C) The specimen type ( *e.g.,* saliva, capillary whole blood).(D) Assay steps and technology used. (E) Specification of required ancillary reagents, instrumentation, and equipment. (F) Specification of the specimen collection, processing, storage, and preparation methods. (G) 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. (H) Information pertaining to the probability of test failure ( *i.e.,* percentage of tests that failed quality control) based on data from clinical samples, a description of scenarios in which a test can fail (*i.e.,* low sample volume, low DNA concentration, etc.), how users will be notified of a test failure, and the nature of followup actions on a failed test to be taken by the user and the manufacturer.(I) Specification of the criteria for test result interpretation and reporting. (J) Information that demonstrates the performance characteristics of the test, including: ( *1* ) Accuracy of study results for each claimed specimen type.( *i* ) Accuracy of the test shall 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 shall 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 shall mimic clinical specimens as much as is feasible and provide an unbiased evaluation of the device accuracy.( *ii* ) 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 device must be predefined and appropriate to the device's intended use. Detailed study protocols must be provided.( *iii* ) Test specimens 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 using either the minimum numbers of samples identified in this paragraph or, when determined appropriate and identified by FDA, a minimum number of samples determined using an alternative method. When appropriate, the same samples may be used in testing to demonstrate the accuracy of testing for multiple genotypes by generating sequence information at multiple relevant genetic locations. At least 20 unique samples representing the wild-type genotype must be tested. To test samples that are heterozygous for the reported variant(s), common variants (>0.1 percent variant frequency in the relevant population) must be tested with at least 20 unique samples. Rare variants (≤0.1 percent variant frequency in the relevant population) must be tested with at least three unique samples. To test samples that are homozygous for the reported variant(s), variants with ≥2 percent variant frequency in a relevant population must be tested with at least 20 unique samples. Variants with a frequency in the relevant population <2 percent and ≥0.5 percent must be tested with at least 10 unique samples. Variants with a frequency in the relevant population <0.5 percent must be tested with at least three unique samples. If variants with a frequency of <0.5 percent are not found within the relevant population and homozygous samples are not tested, then the test results for this homozygous rare variant must not be reported to the user.( *iv* ) Information about the accuracy study shall include the number and type of samples that were compared to bidirectional Sanger sequencing or other methods identified as appropriate by FDA. This information must either be reported in tabular format and arranged by clinically relevant variants or reported using another method identified as appropriate by FDA. As an example, for samples with different genotypes DD, Dd, and dd, the following table represents data from the accuracy study presented in tabular format: ( *v* ) The accuracy represents the degrees of agreement between the device results and the comparator results. The accuracy must be evaluated by measuring different percent agreements (PA) of device results with the comparator results and percent of 'no calls' or 'invalid calls.' Calculate the rate of 'no calls' and 'invalid calls' for each comparator output as %Inv(DD) = A4 /NDD, %Inv(Dd) = B4 /NDd , %Inv(dd) = C4 /Ndd . If 'no calls' or 'invalid calls' are required to be retested according to the device instructions for use, the percent of final 'no calls' or 'invalid calls' must be provided. In the table presenting the results of the accuracy study, use only the final results (*i.e.,* after retesting the initial 'no calls' or 'invalid calls', if required according to the instructions for use). Samples that resulted in a 'no call' or 'invalid call' after retesting must not be included in the final calculations of agreement. If the percentages of 'no calls' or 'invalid calls' for each comparator output are similar, combine these estimates as (A4 + B4 + C4 )/(NDD + NDd + Ndd ) and provide a 95 percent two-sided confidence interval. The percent of final 'no calls' or 'invalid calls' must be clinically acceptable.( *vi* ) Point estimates of percent agreement 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 excluding 'no calls' or 'invalid calls'. The calculations must be performed as follows: ( *vii* ) For percent agreements for DD, Dd and dd (PA(DD|DD), PA(Dd|Dd) and PA(dd|dd)) as described in paragraph (b)(3)(iii)(J)(*1* )(*vi* ) of this section, the 95 percent two-sided confidence intervals must be provided. The accuracy point estimates for percent agreements for DD, Dd and dd must be ≥99 percent per reported variant and overall. Any variants that have a point estimate for either PA(DD|DD), PA(Dd|Dd), or PA(dd|dd) of <99 percent compared to bidirectional sequencing or other methods identified as appropriate by FDA must not be incorporated into test claims and reports. Accuracy results 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 type and by genotype or must be reported using another method identified as appropriate by FDA (see paragraph (b)(3)(iii)(J)(*1* )(*iv* ) of this section).( *viii* ) Information must be reported on the Technical Positive Predictive Value (TPPV) related to the analytical (technical) performance of the device for genotypes in each relevant subpopulation (*e.g.,* ethnicity, gender, age, geographical location, etc.). TPPV is the percentage of individuals with the genotype truly present among individuals whose test reports indicate that this genotype is present. The TPPV depends on the accuracy measures of percent agreements and on the frequency of the genotypes in the subpopulation being studied. The f(DD) is the frequency of DD and f(Dd) is the frequency of Dd in the subpopulation being studied; TPPV must be calculated as described in paragraphs (b)(3)(iii)(J)(*1* )(*ix* ) through (*xi* ) of this section.( *ix* ) For variants where the point estimates of PA(DD|DD), PA(Dd|Dd) and PA(dd|dd) are less than 100 percent, use these point estimates in TPPV calculations.( *x* ) Point estimates of 100 percent in the accuracy study may have high uncertainty about performance of the test in the population. If these variants are measured using highly multiplexed technology, calculate the random error rate for the overall device. The accuracy study described in paragraph (b)(3)(iii)(J) of this section in those cases is more to determine that there is no systematic error in such devices. In those cases, incorporate that rate in the estimation of the percent agreements as calculated in paragraph (b)(3)(iii)(J)(*1* )(*vi* ) of this section and include it in TPPV calculations.( *xi* ) The TPPV for subpopulations with genotype frequencies of f(dd), f(Dd) and f(DD) = 1−f(dd)−f(Dd) in the subpopulation is calculated as: ( *2* ) 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 either include specimens from the claimed sample type (*e.g.,* saliva) representing all genotypes for each variant (*e.g.,* wild type, heterozygous, and homozygous) or, if an alternative panel composition of specimens is identified by FDA as appropriate, a panel composed of those specimens FDA identified as appropriate. A detailed study protocol must be created in advance of the study and must include predetermined acceptance criteria for performance results. The percentage of samples that failed quality control 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 shall report the variants tested in the study and the number of replicates for each variant, and what conditions were tested (*i.e.,* number of runs, days, instruments, reagent lots, operators, specimens/type, etc.). Results must be evaluated and presented in tabular format and stratified by study parameter (*e.g.,* by site, instrument(s), reagent lot, operator, and sample variant). The study must include all extraction steps from the claimed specimen type or matrix, unless a separate extraction reproducibility 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 reproducibility 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.( *3* )*Analytical specificity data:* Data must be provided that evaluates the effect of potential endogenous and exogenous interferents on test performance, including specimen extraction and variant detection. Interferents tested must include those reasonably likely to be potentially relevant to the sample type used for the device.( *4* )*Interfering variant data:* Nucleotide mutations that can interfere with the technology must be cited and evaluated. Data must be provided to demonstrate the effect of the interfering variant(s) on the performance of the correct calls. Alternatively, for each suspected interfering mutation for which data is not provided demonstrating the effect of the interfering variant, the manufacturer must identify the suspected interfering variants in the labeling and indicate that the impact that the interfering variants may have on the assay's performance has not been studied by providing a statement that reads “It is possible that the presence of [insert clearly 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.”( *5* )*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.( *6* )*Reagent stability:* The manufacturer must evaluate reagent stability using wild-type, heterozygous, and homozygous samples. Reagent stability data must demonstrate that the reagents maintain the claimed accuracy and reproducibility. Data supporting such claims must be provided.( *7* )*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.(K) Clinical performance summary. ( *1* ) Information to support the clinical performance of each variant reported by the test must be provided.( *2* ) 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.( *i* ) If odds ratios are available, using information about the genotype distribution either among individuals with the target disease absent, or in the general population, or information about the risk variant frequency and odds ratios, the likelihood ratios for the corresponding device results along with 95 percent confidence intervals must be calculated. Using information about pretest risk (π), an estimate of likelihood ratio (LR), and a relationship between post-test risk R as R/(1−R) = LR·π/(1−π), the post-test risk R must be calculated.( *ii* ) When available, likelihood ratios (LR) for different test results must be presented in a tabular format along with references to the source data or using another method identified as appropriate by FDA as stated in paragraph (b)(3)(iii)(K)(2) of this section. When these values are not directly available in published literature, likelihood ratios can be separately calculated along with the 95 percent confidence interval with references to the source data. Note that a minimum requirement for the presence of the variant's effect on the risk is that a corresponding LR is statistically higher than 1 (a lower bound of 95 percent two-sided confidence interval is larger than 1). It means that the post-test risk is statistically higher than the pretest risk (an observed value of the difference between the post-test and pretest risks).(L) Materials that explain the main concepts and terminology used in the test that includes, but is not limited to: ( *1* )*Definitions:* Scientific terms that are used in the test reports.( *2* )*Prepurchase page:* This page must contain information that informs the user about what the test will provide. This includes, but is not limited to, variant information, the condition or disease 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) 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 section must be provided. This opt-in page must be provided for each disease 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* ) Professional guidelines that recommend when genetic testing for the associated target condition is or is not recommended; and( *iv* ) A recommendation to speak with a health care professional, genetic counselor, or equivalent professional before getting the results of the test.( *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 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 on the test results, information about the population to which the variants in the test is most applicable, the meaning of the result(s), other risks factors that contribute to disease, appropriate followup procedures, how the results of the test may affect the user's family, including children, and links to resources that provide additional information.(M) 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. ( *1* ) The test manufacturer must provide a genetic 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.( *2* ) The test manufacturer must perform pre- and post-test user comprehension studies. The comprehension test questions must include directly evaluating a representative sample of the material being presented to the user as described in paragraph (b)(3)(ii) of this section.( *3* ) 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.( *4* ) The user study must meet the following criteria:( *i* ) 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 shall be well defined in the inclusion and exclusion criteria.( *ii* ) All sources of bias must be predefined and accounted for in the study results with regard to both responders and non-responders.( *iii* ) The testing must follow a format where users have limited time to complete the studies (such as an onsite survey format and a one-time visit with a cap on the maximum amount of time that a participant has to complete the tests).( *iv* ) 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 of 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.( *v* ) 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.( *vi* ) 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.( *vii* ) The analysis of the user comprehension results must include results regarding reports that are provided for each gene/variant/ethnicity tested, statistical methods used to analyze all data sets, and 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, etc.) for each study report must be included.(4) The intended use of the device must not include the following indications for use: (i) Prenatal testing; (ii) Determining predisposition for cancer where the result of the test may lead to prophylactic screening, confirmatory procedures, or treatments that may incur morbidity or mortality to the patient; (iii) Assessing the presence of genetic variants that impact the metabolism, exposure, response, risk of adverse events, dosing, or mechanisms of prescription or over-the-counter medications; or (iv) Assessing the presence of deterministic autosomal dominant variants.

Related Devices

Submission Summary (Full Text)

{0}------------------------------------------------ # EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR The 23andMe Personal Genome Service (PGS) Genetic Health Risk Test for Hereditary Thrombophilia, Alpha-1 Antitrypsin Deficiency, Alzheimer's Disease, Parkinson's Disease, Gaucher Disease Type 1, Factor XI Deficiency, Celiac Disease, G6PD Deficiency, Hereditary Hemochromatosis and Early-Onset Primary Dystonia # DECISION SUMMARY Correction Date: November 2, 2017 This Decision Summary contains corrections to the May 2, 2017 Revised Decision Summary. The Decision Summary originally issued April 6, 2017. ### A. DEN Number: DEN160026 ### B. Purpose for Submission: De Novo request for the 23andMe Personal Genome Service (PGS) Genetic Health Risk Test for Hereditary Thrombophilia, Alpha-1 Antitrypsin Deficiency, Alzheimer's Disease, Parkinson's Disease, Gaucher Disease Type 1, Factor XI Deficiency, Celiac Disease, G6PD Deficiency, Hereditary Hemochromatosis and Early-Onset Primary Dystonia ## C. Measurands: Genomic DNA obtained from a human saliva sample # D. Type of Test: The 23andMe PGS Genetic Health Risk Report for Hereditary Thrombophilia determines and interprets if a person has variants associated with a higher risk of developing harmful blood clots. This report is based on a qualitative genetic test for single nucleotide polymorphism detection of Factor V Leiden variant in the F5 gene (rs6025) and Prothrombin G20210A variant in the F2 gene (rs1799963/i3002432) by using the 23andMe Personal Genome Service. The 23andMe PGS Genetic Health Risk Report for Alpha-1 Antitrypsin Deficiency (AATD) determines if a person has variants associated with a higher risk of developing AATD associated lung or liver disease. This report is based on a qualitative genetic test for single nucleotide polymorphism detection of the PI*Z (rs28929474) and PI*S (rs17580) variants in the SERPINA1 gene by using the 23andMe Personal Genome Service. The 23andMe PGS Genetic Health Risk Report for Late-Onset Alzheimer's Disease determines if a person has variants associated with a higher risk of late-onset Alzheimer's disease. This report is based on a qualitative genetic test for single nucleotide polymorphism {1}------------------------------------------------ detection of s4 (rs429358) variant in the APOE gene by using the 23andMe Personal Genome Service. The 23andMe PGS Genetic Health Risk Report for Parkinson's Disease determines if a person has variants associated with a higher risk of Parkinson's Disease. This report is based on a qualitative genetic test for single nucleotide polymorphism detection of G2019S (rs34637584) variant in the LRRK2 gene and N370S (rs76763715) variant in the GBA gene by using the 23andMe Personal Genome Service. The 23andMe PGS Genetic Health Risk Report for Gaucher Disease Type 1 determines if a person has variants associated with a higher risk of Gaucher Disease. This report is based on a qualitative genetic test for single nucleotide polymorphism detection of the N370S (rs76763715) variant, 84GG (rs387906315) variant and V394L (rs80356769) variant in the GBA gene by using the 23andMe Personal Genome Service. The 23andMe PGS Genetic Health Risk Report for Factor XI Deficiency determines if a person has variants associated with a higher risk of developing Factor XI Deficiency. This report is based on a qualitative genetic test for single nucleotide polymorphism detection of the F283L (rs121965064), E117X (rs121965063), and IVS14+1G>A (rs373297713) variants in the FXI gene by using the 23andMe Personal Genome Service. The 23andMe PGS Genetic Health Risk Report for Celiac disease determines if a person has variants associated with a higher risk of developing Celiac disease. This report is based on a qualitative genetic test for single nucleotide polymorphism detection of the HLA-DQ2.5 haplotype (rs2187668) in the HLA-DQA1 gene by using the 23andMe Personal Genome Service. The 23andMe PGS Genetic Health Risk Report for Glucose-6-Phosphate-Dehydrogenase Deficiency determines if a person has variants associated with a higher risk of developing G6PD Deficiency and a higher risk for episodes of anemia. This report is based on a qualitative genetic test for single nucleotide polymorphism detection of the Val68Met (rs1050828) variant in the G6PD gene by using the 23andMe Personal Genome Service. The 23andMe PGS Genetic Health Risk Report for Hereditary Hemochromatosis determines if a person has variants associated with hereditary hemochromatosis and a higher risk of developing iron overload. This report is based on a qualitative genetic test for the C282Y (rs1800562) and H63D (rs1799945) variants in the HFE gene by using the 23andMe Personal Genome Service. The 23andMe PGS Genetic Health Risk Report for Early-Onset Primary Dystonia determines if a person has variants associated with a higher risk of developing Early-Onset Primary Dystonia. This report is based on a qualitative genetic test for 3 base pair nucleotide deletion (rs724159981) called the deltaE302/303 variant in the DYT1 gene by using the 23andMe Personal Genome Service. # E. Applicant: {2}------------------------------------------------ 23andMe. Inc. # F. Proprietary and Established Names: 23andMe Personal Genome Service # G. Regulatory Information: - 1. Regulation section: 21 CFR 866.5950 - 2. Classification: Class II - 3. Product code(s): PTA - 4. Panel: Immunology ### H. Indications for use: - 1. Indications for use: The 23andMe Personal Genome Service (PGS) Test uses qualitative genotyping to detect the following clinically relevant variants in genomic DNA isolated from human saliva collected from individuals ≥18 years with the Oragene Dx model OGD-500.001 for the purpose of reporting and interpreting Genetic Health Risks (GHR): The 23andMe PGS Genetic Health Risk Report for Hereditary Thrombophilia is indicated for reporting of the Factor V Leiden variant in the F5 gene, and the Prothrombin G20210A variant in the F2 gene. This report describes if a person has variants associated with a higher risk of developing harmful blood clots, but it does not describe a person's overall risk of developing harmful blood clots. This test is most relevant for people of European descent. The 23andMe PGS Genetic Health Risk Report for Alpha-1 Antitrypsin Deficiency is indicated for reporting of the PI*Z and PI*S variants in the SERPINA1 gene. This report describes if a person has variants associated with AAT deficiency and a higher risk for lung or liver disease, but it does not describe a person's overall risk of developing lung or liver disease. This test is most relevant for people of European descent. {3}------------------------------------------------ The 23andMe PGS Genetic Health Risk Report for Late-onset Alzheimer's Disease is indicated for reporting of the £4 variant in the APOE gene. The report describes if a person's genetic result is associated with an increased risk of developing Late-onset Alzheimer's Disease, but it does not describe a person's overall risk of developing Alzheimer's Disease. The £4 variant included in this report is found and has been studied in many ethnicities. Detailed risk estimates have been studied the most in people of European descent. The 23andMe PGS Genetic Health Risk Report for Parkinson's Disease is indicated for reporting of the G2019S variant in the LRRK2 gene and the N370S variant in the GBA gene. The report describes if a person's genetic result is associated with an increased risk of developing Parkinson's disease, but it does not describe a person's overall risk of developing Parkinson's disease. The test is most relevant for people of European, Ashkenazi Jewish, and North African Berber descent. The 23andMe PGS Genetic Health Risk Report for Gaucher Disease Type 1 is indicated for reporting of the N370S, 84GG, and V394L variants in the GBA gene. This report describes if a person has variants associated with an increased risk for developing symptoms of Gaucher Disease Type 1, but it does not describe a person's overall risk of developing Gaucher Disease Type 1. This test is most relevant for people of Ashkenazi Jewish descent. The 23andMe PGS Genetic Health Risk Report for Factor XI Deficiency is indicated for reporting of the variants F283L. E117X. IVS14+1G>A in the F11 gene. This report describes if a person has a variant associated with Factor XI deficiency and the potential for a higher risk of excessive bleeding following trauma or surgery, but it does not describe a person's overall risk for excessive bleeding. This test is most relevant for people of Ashkenazi Jewish descent. The 23andMe PGS Genetic Health Risk Report for Celiac Disease is indicated for reporting of a variant associated with the HLA-DQ2.5 haplotype. The report describes if a person has a haplotype associated with an increased risk of developing celiac disease, but it does not describe a person's overall risk for developing celiac disease. This report is most relevant for people of European descent. The 23andMe PGS Genetic Health Risk Report for Glucose-6-Phosphate-Dehydrogenase Deficiency is indicated for reporting of the Val68Met variant in the G6PD gene. This report describes if a person has a variant associated with G6PD deficiency and a higher risk for episodes of anemia, but it does not describe a person's overall risk of developing anemia. This test is most relevant for people of African descent. The 23andMe PGS Genetic Health Risk Report for Hereditary Hemochromatosis is indicated for reporting of the C282Y and H63D variants in the HFE gene. This report describes if a person has variants associated with hereditary hemochromatosis and a higher risk for iron overload, but it does not describe a person's overall risk of developing iron overload. This report is most relevant for people of European descent. {4}------------------------------------------------ The 23andMe PGS Genetic Health Risk Report for Early-Onset Primary Dystonia (DYT1/TOR1A-Related) is indicated for reporting of the deltaE302/303 variant in the DYT1 gene. This report describes if a person has variants associated with a higher risk for early-onset primary dystonia, but it does not describe a person's overall risk of developing dystonia. This report is most relevant for people of Ashkenazi Jewish descent. - 2. Special conditions for use statements: - For over-the-counter (OTC) use. a. - This test is not a substitute for visits to a healthcare provider. It is recommended that b. you consult with a healthcare provider if you have any questions or concerns about vour results. - c. The 23andMe PGS Genetic Health Risk Tests for Hereditary Thrombophilia, Alpha-1 Antitrypsin Deficiency, Alzheimer's disease, Parkinson's disease, Gaucher Disease, Factor XI Deficiency, Celiac disease, and Glucose-6-Phosphate-Dehydrogenase Deficiency, Early-Onset Primary Dystonia and Hereditary Hemochromatosis do not detect all genetic variants associated with the aforementioned diseases. The absence of a variant tested does not rule out the presence of other genetic variants that may be disease-related. - d. The test is intended for users ≥ 18 years old. - The test does not diagnose any specific health conditions. Results should not be used e. to make medical decisions. - f. 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%. - g. A user's race, ethnicity, age, and sex may affect how the genetic test results are interpreted. - h. Subject to meeting the limitations contained in the special controls under regulation 21 CFR 866.5950. # 3. Special instrument requirements: The 23andMe PGS Genetic Health Risk Tests for Hereditary Thrombophilia. Alpha-1 Antitrypsin Deficiency, Alzheimer's disease, Parkinson's disease, Gaucher Disease Type I. Factor XI Deficiency. Celiac Disease, and Glucose-6-Phosphate-Dehydrogenase Deficiency, Early-Onset Primary Dystonia and Hereditary Hemochromatosis are to be performed using the Tecan Evo and Illumina iScan instruments. GenomeStudio is a modular software application that is used to view and analyze genotypic data obtained from the iScan. Coregen software conducts a variety of control checks on the file, resulting in a final genotype profile for each sample. These data are used to generate test reports on a user's genotype and associated risk of disease. # I. Device Description: {5}------------------------------------------------ The 23andMe PGS is a currently-marketed, non-invasive genetic information service that combines qualitative genotyping data covering genetic ancestry, traits, and certain heritable health conditions from a single multiplex assay with descriptive information derived from peer reviewed, published genetic research studies. It is a direct-toconsumer, over-the-counter, non-diagnostic, DNA genetic testing service intended to provide information and tools for individual users. A user's saliva is 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. Once the sample is collected, it is shipped to one of two Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories for processing. DNA is isolated from the saliva and tested in a multiplex assay using a customized genotyping chip and instrumentation manufactured by Illumina. The multiplex assay simultaneously tests for more than 500,000 variants, including those for the indications proposed herein. The raw data is generated using Illumina GenomeStudio software, and then sent to 23andMe (the Manufacturer). The data are analyzed using the Manufacturer's proprietary Coregen software, and a genotype is determined for each tested variant. The results for certain of these variants are used to generate personalized reports for users that provide information about the diseases associated with the detected variant. Personalized reports are generated for each user that provide results of the testing performed. These reports tell the user which variant(s) has/have been detected in their sample and provide information on the risk of 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. The reports provide scientifically valid information about the risks associated with the presence of a particular variant. The reports are designed to help users understand the meaning of their results and any appropriate actions that may be taken based on their results. ### J. Substantial Equivalence Information: - 1. Predicate device name: No predicate device exists. - 2. Predicate 510(k) number: Not applicable. - 3. Comparison with predicate: Not applicable. {6}------------------------------------------------ ## K. Standard/Guidance Document Referenced: Not applicable. ### L. Test Principle: The PGS is indicated to be performed using the BeadChip v4 assay (Illumina Infinium HumanOmniExpress-24 format chip), which covers more than 500,000 genetic markers. The BeadChip 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 variant-specific primers, followed by extension with hapten-labeled nucleotides. The primers hybridize adjacent to the variants 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 pre-defined report templates specific for each condition associated with each genotype. Genotypes are determined using the GenomeStudio and Coregen software packages. ### M. Performance Characteristics: Saliva samples were collected using the Oragene Dx saliva collection device (OGD-500.001). The samples were tested on the Illumina Infinium BeadChip. Results were analyzed using the Illumina iScan System and GenomeStudio and Coregen software. - 1. Analytical performance: The results of all the analytical performance studies met the pre-determined acceptance criteria. - a. Precision/Reproducibility ### Reproducibility for Variants Associated with Hereditary Thrombophilia Reproducibility studies were conducted for the two genes associated with hereditary thrombophilia: Factor V Leiden and Prothrombin G20210A. The reproducibility studies were designed to determine the imprecision due to assay run, lot, instrument, operator, day, and site. Three Factor V Leiden common (wild type) DNA samples, two Factor V heterozygous DNA samples, one Factor V Leiden homozygous rare DNA sample, three Prothrombin G20210A common (wild type) DNA samples, one Prothrombin G20210A heterozygous DNA sample, and one Prothrombin G20210A homozygous rare DNA sample were included in the precision /reproducibility studies. Genotypes of these cell line samples were confirmed through bidirectional Sanger sequencing. These samples were genotyped by the PGS Test in a blinded fashion. {7}------------------------------------------------ Samples were genotyped by the PGS Test using multiple lots of reagents, three operator teams, and nine different combinations of two instrument types (Tecan ([three instruments] and iScan [3 instruments]), over the course of five days, at two independent laboratory sites. This precision study yielded 100% correct genotype calls for all samples with a valid call across multiple days, operator teams, instruments, and reagent lots at both laboratory sites. Information regarding samples that failed quality control (FQC) was also evaluated. ### Factor V Leiden The combined data of the reproducibility study is presented in the table below: | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of<br>FQCs | Percentage<br>of<br>FQCs | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|----------------------|--------------------------| | CC<br>(homozygous common) | 1080 | 1061 | 0 | 19 | 1.76% | | CT (heterozygous) | 540 | 533 | 0 | 7 | 1.30% | | TT (homozygous rare) | 270 | 258 | 0 | 12 | 4.44% | | Total | 1890 | 1852 | 0 | 38 | 2.01% | Genotyping results produced 98.0 % (1852/1890) replicates that were called correctly and 2.01% (38/1890) replicates that did not pass quality control (QC) acceptance criteria. Samples with FQC on the first run are re-tested per laboratory standard operating procedures (SOPs); an anticipated rate of samples with two FQCs based on the reproducibility study data of cell line samples is 0.04% (0.020 x 0.020). | Site 1 | | | | | | |------------------------------|------------------------------------------------|----------------------------|------------------------------|----------------------|--------------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct Calls | Number of<br>Incorrect Calls | Number<br>of<br>FQCs | Percentage<br>of<br>FQCs | | CC<br>(homozygous<br>common) | 540 | 537 | 0 | 3 | 0.56% | | CT<br>(heterozygous) | 270 | 268 | 0 | 2 | 0.74% | | TT<br>(homozygous<br>rare) | 135 | 135 | 0 | 0 | 0.00% | # Results for Factor V Leiden stratified by site and genotype (all valid calls were 100% correct at both sites): {8}------------------------------------------------ | Total | 945 | 940 | 0 | 5 | 0.53% | |-------|-----|-----|---|---|-------| |-------|-----|-----|---|---|-------| | Site 2 | | | | | | |------------------------------|------------------------------------------------|----------------------------|------------------------------|----------------------|--------------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct Calls | Number of<br>Incorrect Calls | Number<br>of<br>FQCs | Percentage<br>of<br>FQCs | | CC<br>(homozygous<br>common) | 540 | 524 | 0 | 16 | 2.96% | | CT<br>(heterozygous) | 270 | 265 | 0 | 5 | 1.85% | | TT<br>(homozygous<br>rare) | 135 | 123 | 0 | 12 | 8.89% | | Total | 945 | 912 | 0 | 33 | 3.49% | #### Prothrombin G20210A The combined data of the reproducibility study of Prothrombin G20210A is presented in the table below: | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of<br>FQCs | Percentage<br>of<br>FQCs | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|----------------------|--------------------------| | GG (homozygous<br>common) | 1080 | 1060 | 0 | 20 | 1.85% | | AG (heterozygous) | 270 | 262 | 0 | 8 | 2.96% | | AA (homozygous<br>rare) | 270 | 268 | 0 | 2 | 0.74% | | Total | 1620 | 1590 | 0 | 30 | 1.85% | Genotyping results produced 98.1 % (1590/1620) replicates that were called correctly and 1.85% (30/1620) replicates that did not pass QC acceptance criteria. Samples with FQC on the first run are re-tested per laboratory SOPs; therefore, an anticipated rate of samples with two FQCs based on the reproducibility study data of cell line samples is 0.03% (0.0185 x 0.0185). # Results for Prothrombin G2010A stratified by site and genotype (all valid calls were 100% correct at both sites): {9}------------------------------------------------ | Site 1 | | | | | | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|----------------------|--------------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of<br>FQCs | Percentage<br>of<br>FQCs | | GG (homozygous<br>common) | 540 | 539 | 0 | 1 | 0.19% | | AG (heterozygous) | 135 | 132 | 0 | 3 | 2.22% | | AA (homozygous<br>rare) | 135 | 134 | 0 | 1 | 0.74% | | Total | 810 | 805 | 0 | 5 | 0.62% | | Site 2 | | | | | | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|----------------------|--------------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of<br>FQCs | Percentage<br>of<br>FQCs | | GG (homozygous<br>common) | 540 | 521 | 0 | 19 | 1.48% | | AG (heterozygous) | 135 | 130 | 0 | 5 | 3.7 | | AA (homozygous<br>rare) | 135 | 134 | 0 | 1 | 0.74% | | Total | 810 | 785 | 0 | 25 | 3.09% | ### Reproducibility for Variants Associated with Alpha-1 Antitrypsin Deficiency Reproducibility studies were conducted for the two variants associated with Alpha-1 Antitrypsin Deficiency: PI*Z and PI*S in the SERPINA1 gene. Two PI*Z SERPINA1 homozygous common DNA samples, one heterozygous PI*Z SERPINA1 DNA sample, one PI*Z SERPINA1 homozygous rare DNA sample, two PI*S SERPINA1 homozygous common DNA samples, one heterozygous PI*S SERPINA1 DNA sample, and one PI*S SERPINA1 homozygous rare DNA sample were included in the reproducibility study. The study design and data analysis for Alpha-1 Antitrypsin Deficiency was identical to the Hereditary Thrombophilia reproducibility study reported above. ### PI*Z SERPINA1 {10}------------------------------------------------ | Genotypee | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of FQCs | Percentage of<br>FQCs | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------| | CC (homozygous<br>common) | 810 | 797 | 0 | 13 | 1.60% | | CT (heterozygous) | 270 | 261 | 0 | 9 | 3.33% | | TT (homozygous<br>rare) | 270 | 270 | 0 | 0 | 0% | | Total | 1350 | 1328 | 0 | 22 | 1.63% | The combined data of the reproducibility study of PI*Z SERPINA1 is presented in the table below Genotyping results produced 98.4 % (1328/1350) replicates that were called correctly and 1.63% (22/1350) replicates that did not pass QC acceptance criteria. Samples with FQC on the first run are re-tested per laboratory SOPs; therefore, an anticipated rate of samples with two FQCs based on the reproducibility study data of cell line samples is 0.03% (0.0163 x 0.0163). | Site 1 | | | | | | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of FQCs | Percentage<br>of FQCs | | CC (homozygous<br>common) | 405 | 404 | 0 | 1 | 0.25% | | CT (heterozygous) | 135 | 131 | 0 | 4 | 2.97% | | TT (homozygous<br>rare) | 135 | 135 | 0 | 0 | 0.00% | | Total | 675 | 670 | 0 | 5 | 0.74% | Results for P1*Z SERPINA1 stratified by site and genotype (all valid calls were 100% correct at both sites): | Site 2 | | | | | | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------| | Genotpye | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of FQCs | Percentage<br>of FQCs | | CC (homozygous<br>common) | 405 | 393 | 0 | 12 | 2.96% | | CT (heterozygous) | 135 | 130 | 0 | 5 | 3.70% | | TT (homozygous<br>rare) | 135 | 135 | 0 | 0 | 0.00% | {11}------------------------------------------------ | Site 2 | | | | | | |----------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------| | Genotpye | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of FQCs | Percentage<br>of FQCs | | Total | 675 | 658 | 0 | 17 | 2.52% | # PI*S SERPINA1 The combined data of the reproducibility study of PI*S SERPINA1 is presented in the table below. | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of FQCs | Percentage of<br>FQCs | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------| | TT (homozygous<br>common) | 810 | 797 | 0 | 13 | 1.60% | | AT (heterozygous) | 270 | 269 | 0 | 1 | 0.37% | | AA (homozygous<br>rare) | 225 | 216 | 0 | 9 | 4.00% | | Total | 1305 | 1282 | 0 | 23 | 1.76% | Genotyping results produced 98.2 % (1282/1305) replicates that were called correctly and 1.76% (23/1305) replicates that did not pass QC acceptance criteria. Samples with FQC on the first run are re-tested per laboratory SOPs; therefore, an anticipated rate of samples with two FQCs based on the reproducibility study data of cell line samples is 0.03% (0.0176 x 0.0176). # Results for P1*S SERPINA1 stratified by site and genotype (all valid calls were 100% correct at both sites): | Site 1 | | | | | | | |----------------------------------------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------|--| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of FQCs | Percentage of<br>FQCs | | | TT (homozygous<br>common) 2<br>samples | 405 | 404 | 0 | 1 | 0.25% | | | AT (heterozygous)<br>1 sample | 135 | 135 | 0 | 0 | 0.00% | | | AA (homozygous<br>rare) 1 sample | 135 | 133 | 0 | 2 | 1.48% | | | Total | 675 | 672 | 0 | 3 | 0.44% | | {12}------------------------------------------------ | Genotpye | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number<br>of<br>FQCs | Percentage of<br>FQCs | |----------------------------------------|------------------------------------------------|-------------------------------|---------------------------------|----------------------|-----------------------| | TT (homozygous<br>common) 2<br>samples | 405 | 393 | 0 | 12 | 2.96% | | AT (heterozygous)<br>1 sample | 135 | 134 | 0 | 1 | 0.74% | | AA (homozygous<br>rare) 1 sample | 90 | 83 | 0 | 7 | 7.78% | | Total | 630 | 610 | 0 | 20 | 3.17% | # Reproducibility for the Variant Associated with Late-onset Alzheimer's Disease Reproducibility studies were conducted for the APOE s4 variant associated with Late-onset Alzheimer's Disease. Three APOE &4 homozygous common DNA samples, two heterozygous APOE &4 DNA samples, and one APOE &4 homozygous rare DNA sample were included in the reproducibility study. These DNA samples were genotyped by the PGS Test in a blinded fashion, using three lots of reagents, three operator teams per day, and nine different combinations of two instrument types (Tecan [3 instruments] and iScan [3 instruments]), over the course of three days, at each of two independent laboratory sites. The combined data of the reproducibility study of APOE &4 is presented in the table below. | Genoptye | Number of Replicates (including FQCs) | Number of Correct Calls | Number of Incorrect Calls | Number of FQCs | Percentage of FQCs | |------------------------|---------------------------------------|-------------------------|---------------------------|----------------|--------------------| | TT (homozygous common) | 486 | 477 | 0 | 9 | 1.89% | | CT (heterozygous) | 324 | 318 | 0 | 6 | 1.89% | | CC (homozygous rare) | 162 | 162 | 0 | 0 | 0.00% | | Total | 972 | 957 | 0 | 15 | 1.54% | Genotyping results produced 98.5 % (957/972) replicates that were called correctly and 1.54% (15/972) replicates that did not pass OC acceptance criteria. Samples with FQC on the first run are re-tested per laboratory SOPs; therefore, an anticipated rate of samples with two FQCs based on the precision study data of cell line samples is 0.02% (0.0154x0.0154). {13}------------------------------------------------ # Results for APOE &4 stratified by site and genotype (all valid calls were 100% correct at both sites): | Site 1 | | | | | | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | | TT (homozygous<br>common) | 243 | 242 | 0 | 1 | 0.41% | | CT (heterozygous) | 162 | 162 | 0 | 0 | 0.00% | | CC (homozygous<br>rare) | 81 | 81 | 0 | 0 | 0.00% | | Total | 486 | 485 | 0 | 1 | 0.21% | | Site 2 | | | | | | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------| | Sample | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | | TT (homozygous<br>common) | 243 | 235 | 0 | 8 | 3.40% | | CT (heterozygous) | 162 | 156 | 0 | 6 | 3.85% | | CC (homozygous<br>rare) | 81 | 81 | 0 | 0 | 0.00% | | Total | 486 | 472 | 0 | 14 | 2.97% | # Reproducibility for Variants Associated with Parkinson's Disease: Reproducibility studies were conducted for two variants associated with Parkinson's Disease: G2019S in the LRRK2 gene and N370S in the GBA gene. Two G2019S LRRK2 homozygous common DNA samples, one heterozygous G2019S LRRK2 DNA sample, one homozygous rare G2019S LRRK2 DNA sample, two N370S GBA homozygous common DNA samples, three heterozygous N370S GBA DNA samples, and one homozygous rare N370S GBA DNA samples were included in the reproducibility study. The study design and data analysis for Parkinson's Disease was identical to the Hereditary Thrombophilia precision study reported above. # G2019S LRRK2 {14}------------------------------------------------ | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------| | GG (homozygous<br>common) | 324 | 315 | 0 | 9 | 2.77% | | AG (heterozygous) | 162 | 161 | 0 | 1 | 0.62% | | AA (homozygous<br>rare) | 162 | 162 | 0 | 0 | 0.00% | | Total | 648 | 638 | 0 | 10 | 1.54% | The combined data of the reproducibility study of G2019S LRRK2 is presented in the table below: Genotyping results produced 98.5 % (638/648) replicates that were called correctly and 1.54% (10/648) replicates that did not pass QC acceptance criteria. Samples with FQC on the first run are re-tested per laboratory SOPs; therefore, an anticipated rate of samples with two FQCs based on the precision study data of cell line samples is 0.02% (0.0154 x 0.0154). # Results for G2109S LRRK2 stratified by site and genotype (all valid calls were 100% correct at both sites): | Site 1 | | | | | | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | | GG (homozygous<br>common) | 162 | 161 | 0 | 1 | 0.62% | | AG (heterozygous) | 81 | 81 | 0 | 0 | 0.00 | | AA (homozygous<br>rare) | 81 | 81 | 0 | 0 | 0.00 | | Total | 324 | 323 | 0 | 1 | 0.31% | | Site 2 | | | | | | |---------------------------|------------------------------------------------|-------------------------------|---------------------------------|-------------------|-----------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | | GG (homozygous<br>common) | 162 | 154 | 0 | 8 | 3.09% | | AG (heterozygous) | 81 | 80 | 0 | 1 | 1.23% | | AA (homozygous<br>rare) | 81 | 81 | 0 | 0 | 0.00% | | Total | 324 | 315 | 0 | 9 | 2.78% | {15}------------------------------------------------ # N370S GBA The combined data of the reproducibility study of N370S GBA is presented in the table below: | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number<br>of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | |---------------------------|------------------------------------------------|-------------------------------|------------------------------------|-------------------|-----------------------| | TT (homozygous<br>common) | 324 | 314 | 0 | 10 | 3.09% | | CT (heterozygous) | 486 | 466 | 0 | 20 | 4.12% | | CC (homozygous<br>rare) | 162 | 155 | 0 | 7 | 4.32% | | Total | 972 | 935 | 0 | 37 | 3.81% | Genotyping results produced 96.2 % (935/972) replicates that were called correctly and 3.81% (37/972) replicates that did not pass OC acceptance criteria. Samples with FQC on the first run are re-tested per laboratory SOPs; therefore, an anticipated rate of samples with two FQCs based on the reproducibility study data of cell line samples is 0.15% (0.0381 x 0.0381). # Results for N370S GBA stratified by site and genotype (all valid calls were 100% correct at both sites): | Site 1 | | | | | | |----------------------------------------|------------------------------------------------|-------------------------------|------------------------------------|-------------------|-----------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number<br>of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | | TT (homozygous<br>common) 2<br>samples | 162 | 158 | 0 | 4 | 1.23% | | CT (heterozygous)<br>3 samples | 243 | 232 | 0 | 11 | 4.53% | | CC (homozygous<br>rare) 1 sample | 81 | 77 | 0 | 4 | 4.94% | | Total | 486 | 467 | 0 | 19 | 3.91% | | Site 2 | | | | | | |---------------------------|------------------------------------------------|-------------------------------|------------------------------------|-------------------|-----------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number<br>of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | | TT (homozygous<br>common) | 162 | 156 | 0 | 6 | 3.70% | {16}------------------------------------------------ | Site 2 | | | | | | |-------------------------|------------------------------------------------|-------------------------------|------------------------------------|-------------------|-----------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number<br>of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | | CT (heterozygous) | 243 | 234 | 0 | 9 | 3.70% | | CC (homozygous<br>rare) | 81 | 78 | 0 | 3 | 3.70% | | Total | 486 | 468 | 0 | 18 | 3.70% | # Reproducibility for the Variant Associated with Gaucher Disease Type I Reproducibility studies were conducted for the N370S variant in the GBA gene associated with Gaucher Disease Type I. Two N370S GBA homozygous common DNA samples, three heterozygous N370S GBA DNA samples, and one homozygous rare N370S GBA DNA sample were included in the reproducibility study. The study design and data analysis for Gaucher Disease was identical to the Alzheimer's Disease study reported above. # N370S GBA The combined data of the reproducibility study of N370S GBA is presented in the table below: | Genotypee | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number<br>of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | |---------------------------|------------------------------------------------|-------------------------------|------------------------------------|-------------------|-----------------------| | TT (homozygous<br>common) | 525 | 510 | 0 | 15 | 2.86% | | CT (heterozygous) | 784 | 768 | 0 | 26 | 3.32% | | CC (homozygous<br>rare) | 262 | 254 | 0 | 8 | 3.05% | | Total | 1571 | 1532 | 0 | 49 | 3.12% | Genotyping results produced 97.5 % (1532/1571) replicates that were called correctly and 3.12% (49/1571) replicates that did not pass OC acceptance criteria. Samples with FQC on the first run are re-tested per laboratory SOPs; therefore, an anticipated rate of samples with two FQCs based on the reproducibility study data of cell line samples is 0.10% (0.0312 x 0.0312). # Results for N370S GBA stratified by site and genotype (all valid calls were 100% correct at both sites): {17}------------------------------------------------ | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number<br>of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | |---------------------------|------------------------------------------------|-------------------------------|------------------------------------|-------------------|-----------------------| | TT (homozygous<br>common) | 264 | 258 | 0 | 6 | 2.27% | | CT (heterozygous) | 394 | 383 | 0 | 11 | 2.79% | | CC (homozygous<br>rare) | 131 | 127 | 0 | 4 | 3.05% | | Total | 789 | 768 | 0 | 21 | 2.66% | | Site 2 | | | | | | |---------------------------|------------------------------------------------|-------------------------------|------------------------------------|-------------------|-----------------------| | Genotype | Number of<br>Replicates<br>(including<br>FQCs) | Number of<br>Correct<br>Calls | Number<br>of<br>Incorrect<br>Calls | Number of<br>FQCs | Percentage<br>of FQCs | | TT (homozygous<br>common) | 261 | 252 | 0 | 9 | 3.45% | | CT (heterozygous) | 390 | 385 | 0 | 15 | 3.85% | | CC (homozygous<br>rare) | 131 | 127 | 0 | 4 | 3.05% | | Total | 782 | 754 | 0 | 28 | 3.58% | The Manufacturer affirmed that reproducibility studies were completed for the variants described in the table below. The study design, acceptance criteria, and results for the reproducibility were the same for each of the variants listed in the table and the same as the studies that were completed for the variants for Hereditary Thrombophilia, Alpha-1 Antitrypsin Deficiency, Alzheimer's Disease, Parkinson's Disease, and Gaucher Disease Type 1 (N370S variant). Each variant was tested with wild-type, heterozygous and homozygous samples, except where indicated. | Disease | Gene | Variant | Precision Completed | |-----------------------------------------------------------|-----------|-------------|---------------------| | Gaucher Disease Type 1 | GBA | 84GGΨ | Yes | | Gaucher Disease Type 1 | GBA | V394LΨ | Yes | | Factor XI Deficiency | Factor XI | F283L | Yes | | Factor XI Deficiency | Factor XI | E117X | Yes | | Factor XI Deficiency | Factor XI | IVS14+1G>AΨ | Yes | | Celiac Disease | HLA-DQA1 | HLA-DQA1 | Yes | | Glucose-6-Phosphate<br>Dehydrogenase Deficiency<br>(G6PD) | G6PD | Val68Met* | Yes | | Hereditary<br>Hemochromatosis | HFE | C282Y | Yes | | Hereditary<br>Hemochromatosis | HFE | H63D | Yes | {18}------------------------------------------------ | Disease | Gene | Variant | Precision Completed | |---------------------------------|------|----------------|---------------------| | Early-Onset Primary<br>Dystonia | DYT1 | deltaE302/303Ψ | Yes | *Hemizygous genotype not included "Homozygous genotype samples were not tested therefore homozygous test results will not be reported to user. - b. Linearity/assay Reportable Range: Not applicable. - Traceability, Stability, Expected Values (controls, calibrators, or methods): C. The PGS requires two types of controls: the sample processing control and the reproducibility control. The control material is genotyped on the 23andMe BeadChip according to routine SOPs at the contracted laboratory sites. Each new lot of the reproducibility control is tested by comparison with reference BeadChip genotype results. The sample processing control is run on every sample genotyping plate and the reproducibility control is run approximately once per week. Historical data from all such runs were analyzed for one lot of the sample processing control spanning three months and one lot of the reproducibility control spanning one year. Stability protocols and acceptance criteria were reviewed and acceptable. 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. - d. Detection Limit: # Detection limit study for variants in the Factor V Leiden, Prothrombin G20210A, SERPINA1, APOE, LRRK2, and GBA variants The Limit of Detection (LoD) study was performed to determine the lowest concentration of DNA that is necessary for successful assigmment of the correct variant using the 23andMe PGS Test. The Manufacturer presented the results of the LoD study for the following genes and variants: Factor V Leiden F5 and Prothrombin G20210A F2 variants for Hereditary Thrombophilia; PI*Z and PI*S in SERPINA1 for Alpha-1 Antitrypsin Deficiency; &4 variant in the APOE gene for Alzheimer's disease; and the G2019S variant in the LRRK2 gene and the N370S variant in the GBA gene for Parkinson's and Gaucher disease. Each sample was diluted to three different DNA concentrations and genotyped by the PGS Test in a blinded fashion using three lots of reagents. To confirm the genotype call, each sample was also sequenced by bidirectional sequencing. Genotype calls from the PGS Test were compared with sequenced genotypes to determine the rates of correct genotype calls at each DNA concentration. The LoD was defined as the lowest DNA concentration at which at least 95% of samples yielded the correct call. {19}------------------------------------------------ The LoD study vielded 100% correct genotype calls for all samples and all reagent lots tested at sample DNA concentrations of 5, 15, and 50 ng/uL. Therefore, the study passed the LoD acceptance criteria at a sample DNA concentration of 5 ng/uL. This LoD study shows that the PGS Test is valid for the variants tested using samples with a DNA concentration between 5 ng/uL to 50 ng/uL. The Manufacturer has claimed a LoD of 15 ng/uL. The Manufacturer affirmed that detection limit studies were completed for the variants described in the table below. The study design, acceptance criteria, and results for the detection limit studies were the same for each of the variants listed in the table and the same as the studies that were completed for the variants for Hereditary Thrombophilia. AATD. Alzheimer's Disease. Parkinson's Disease, and Gaucher Disease, reported above. Each variant was tested with a wild-type, heterozygous, and homozygous sample, except where indicated. | Disease | Gene | Variant | LoD Completed | |-----------------------------------------------------------|-----------|----------------|---------------| | Gaucher Disease Type 1 | GBA | 84GGΨ | Yes | | Gaucher Disease Type 1 | GBA | V394LΨ | Yes | | Factor XI Deficiency | Factor XI | F283L | Yes | | Factor XI Deficiency | Factor XI | E117X | Yes | | Factor XI Deficiency | Factor XI | IVS14+1G>AΨ | Yes | | Celiac Disease | HLA-DQA1 | HLA-DQA1 | Yes | | Glucose-6-Phosphate<br>Dehydrogenase Deficiency<br>(G6PD) | G6PD | Val68Met* | Yes | | Hereditary Hemochromatosis | HFE | C282Y | Yes | | Hereditary Hemochromatosis | HFE | H63D | Yes | | Early-Onset Primary<br>Dystonia | DYT1 | deltaE302/303Ψ | Yes | *Hemizygous genotype not included "Homozygous genotype samples were not tested therefore homozygous test results will not be reported to user. - e. Interfering Substances #### Endogenous and Exogenous substances A series of studies were conducted to assess the effects of endogenous substances, exogenous substances, microbial substances, and smoking on the 23andMe PGS Test. The results of the Endogenous Interference Study can be found in the Decision Summary for DEN140044. The PGS Test requires the use of an FDA cleared collection device (K141410). The cleared device includes instructions for use instructing the user to not eat. drink. smoke, or chew gum for 30 minutes prior to collecting their saliva, thus minimizing the presence of interferents in the sample. The Instructions for Use (IFU) was tested {20}------------------------------------------------ for user comprehension and a paper version of the IFU is included in every collection kit. Should an interfering substance be present after DNA has been extracted and an insufficient concentration or quality of DNA is available, then the sample is managed per standard operating procedures that are pre-determined by the Manufacturer. #### Interfering Mutations Analyses were performed to identify potentially interfering variants within the 50 nucleotide probe-binding region of the variant being detected. If samples for the potentially interfering mutations were not available to test the impact on detection of the corresponding clinically relevant variant, the Manufacturer added a statement in the device labeling stating that the impact of that potentially interfering mutation on the performance of the device has not been evaluated. The results follow for each claimed variant. #### Interfering Mutations for Hereditary Thrombophilia: Two potentially interfering mutations for Factor V Leiden and one potentially interfering mutation for Prothrombin G20210A were identified. For Factor V Leiden the potential interfering mutations include rs760488939 and rs763859650. For Prothrombin G20210A the potential interfering mutation is rs112016113. Interference due to these mutations was not tested. #### Interfering Mutations for Alph-1 Antitrypsin Deficiency: Nine potentially interfering mutations were identified for PI*Z SERPINA1and six potentially interfering mutation in PI*S SERPINA1. The potentially interfering mutations include: rs148362959, rs533419579, rs551595739, rs201774333, rs 143370956, rs 1131139, rs200945035, rs373630097 and rs9630 for PI*Z variant and rs538675821. rs550592374. rs141095970. rs149537225. rs1049800 and rs2230075 for PI*S variant. Interference due to these mutations was not tested. #### Interfering Mutations for Alzheimer's disease: Three potentially interfering mutations were identified for APOEs4. The potential interfering mutations include rs11542041, rs573658040, and rs543363163. Interference due to these mutations was not tested. #### Interfering Mutations for Parkinson's Disease: Two potentially interfering mutations each for LRRK2 and GBA variants were identified. For the G2019S variant for LRRK2 the potential interfering mutations include rs150219613 and rs183394865. For the N370S variant for GBA the potential interfering mutations include rs187143994 and rs111417507. Interference due to these mutations was not tested. #### Interfering Mutations for Gaucher Disease: Seven potentially interfering mutations were identified for GBA. The potentially interfering mutations include: rs104886460, rs143187997 and rs150466109 for the 84GG variant: rs14917112 and rs201499639 for the V394L variant: and rs187143994 and rs111417507 for the N370S variant. Interference due to these mutations was not tested. {21}------------------------------------------------ Interfering Mutations for Factor XI Deficiency: Eight potentially interfering mutations were identified for Factor XI. The potential interfering mutations include rs200218867, rs549554738, rs569456903, and rs147592940 for the F283L variant and rs200593979. rs199657604 and rs34807019 for the E117X variant. Interference due to these mutations was not tested. Interfering Mutations for Celiac Disease: Six potentially interfering mutations were identified for HLA-DOA1. The potential interfering mutations include rs373744062, rs34481484, rs535725525, rs116178934, rs118073417, and rs9272482. Interference due to these mutations was not tested. Interfering Mutations for Glucose-6-Phosphate Dehydrogenase Deficiency: One potentially interfering mutation was identified for Val68Met. The potential interfering mutation is rs138687036. Interference due to this mutation was not tested. Interfering Mutations for Hereditary Hemochromatosis: Seven potentially interfering mutations were identified for Hemochromatosis. The potentially interfering mutations include rs140080192 and rs143175221 for the C282Y variant and rs28934889, rs147297176, rs147426902, rs556335391 and rs62625342 for the H63D variant. Interference due to these mutations was not tested. Interfering Mutations for Early Onset Primary Dystonia: One potentially interfering mutation was identified for ΔE302/303. The potential interfering mutation is rs188191403. Interference due to this mutation was not tested. - Assay Cut-off: f. Not applicable. - g. Specimen Stability at 2-8° C Saliva samples for testing are collected with the Oragene Dx collection device. See K141410 for sample stability information. - h. Shipping Stability Saliva samples are shipped for testing in the Oragene-Dx collection device. See K141410 for sample shipping stability information. # 2. Comparison Studies: - a. Comparison with Sanger Bidirectional Sequencing: Comparison for Hereditary Thrombophilia, Alpha-1 Antitrypsin Deficiency, Alzheimer's Disease, Parkinson's Disease, Gaucher Disease: {22}------------------------------------------------ Accuracy was evaluated through calculation of agreement of the genetic variant determinations between the 23andMe PGS Test and results from Sanger bidirectional sequencing (comparator). All PGS genotyping was performed at an independent laboratory site. Saliva samples were selected from the 23andMe customer biobank based on their predetermined genotype. All chosen samples were then genotyped using Sanger bidirectional sequencing. Genotyping results were compared between the PGS test and bidirectional sequencing to calculate percent agreement (PA) with the sequencing results considered to be "truth." The comparison study for each report is provided below. The accuracy data generated for each test report met the predefined acceptance criteria outlined in the special controls. ### Comparison for Hereditary Thrombophilia: The goal of the comparison studies was to evaluate genotype assignment between the 23andMe PGS Test and the gold standard, Sanger bidirectional sequencing, for the Factor V Leiden variant of the F5 gene and the Prothrombin G20210A variant of the F2 gene. | Factor V Leiden variant of the F5 gene | | | | | |----------------------------------------|-------------------------|---------------------------------|----|----| | | | Sanger Bidirectional Sequencing | | | | | Genotype | CC | CT | TT | | 23andMe<br>PGS Test | CC (homozygous common) | 68 | 0 | 0 | | | CT (heterozygous) | 0 | 68 | 0 | | | TT (homozygous rare) | 0 | 0 | 67 | | | 'no calls' or 'invalid" | 0 | 0 | 0 | | Total | | 68 | 68 | 67 | Percent Agreement (PA) of PGS test results with comparator results of saliva samples: PA (CC|CC) = 100% (68/68) with 95% CI: 94.7% to 100%; PA (CT|CT) = 100% (68/68) with 95% CI: 94.7% to 100%; PA (TT|TT) =100% (67/67) with 95% CI: 94.6% to 100%; Percent of no calls or invalid is 0.0% (0/203) with 95% CI: 0.0% to 1.9%. The minor variant frequency for Factor V Leiden in individuals of European descent reported in published literature is 3%-15%; technical (analytical) positive predictive values for 23andMe PGS test results of CT and TT are ≥ 99.5% and ≥ 99.1% correspondingly. | Prothrombin G20210A variant of the F2 gene | | | | | |--------------------------------------------|-------------------------|---------------------------------|----|----| | | | Sanger Bidirectional Sequencing | | | | | Genotype | GG | AG | AA | | 23andMe<br>PGS Test | GG (homozygous common) | 68 | 0 | 0 | | | AG (heterozygous) | 0 | 67 | 0 | | | AA (homozygous rare) | 0 | 0 | 66 | | | 'no calls' or 'invalid" | 0 | 0 | 0 | {23}------------------------------------------------ | Total | 68 | 67 | 66 | |-------|----|----|----| |-------|----|----|----| ## Percent Agreement (PA) of PGS test results with comparator results of saliva samples: PA (GG|GG) = 100% (68/68) with 95% CI: 94.7% to 100%; PA (AG|AG) = 100% (67/67) with 95% CI: 94.6% to 100%; PA (AA|AA) =100% (66/66) with 95% CI: 94.5% to 100%; Percent of no calls or invalid is 0.0% (0/201 ) with 95% CI: 0.0% to 1.9%. The minor variant frequency for Prothrombin G20210A in individuals of European descent reported in published literature is 1%-3%; technical (analytical) positive predictive values for 23andMe PGS test results of AG and AA are ≥ 98.6% and ≥ 97.2% correspondingly. ### Comparison for Alpha-1 Antitrypsin Deficiency The goal of the comparison studies was to evaluate genotype assignment between the 23andMe PGS Test and a gold standard, Sanger bidirectional sequencing for the PI*S and PI*Z variants of the SERPINA1 gene. The study design and acceptance criteria were identical to the study for Hereditary Thrombophilia. | PI*Z variant of the SERPINA1 gene | | | | | |-----------------------------------|-------------------------|---------------------------------|----|----| | | | Sanger Bidirectional Sequencing | | | | | Genotype | CC | CT | TT | | 23andMe<br>PGS Test | CC (homozygous common) | 70 | 0 | 0 | | | CT (heterozygous) | 0 | 68 | 0 | | | TT (homozygous rare) | 0 | 0 | 69 | | | 'no calls' or 'invalid" | 0 | 0 | 0 | | Total | | 70 | 68 | 69 | ### Percent Agreement (PA) of PGS test results with comparator results of saliva samples: PA (CC|CC) = 100% (70/70) with 95% CI: 94.8% to 100%; PA (CT|CT) = 100% (68/68) with 95% CI: 94.7% to 100%; PA (TT|TT) =100% (69/69) with 95% CI: 94.7% to 100%; Percent of no calls or invalid is 0.0% (0/207) with 95% CI: 0.0% to 1.8%. Minor variant frequency for PI*Z SERPINA1 in individuals of European descent in the 23andMe database was 3.63%; technical (analytical) positive predictive values for 23andMe PGS test result of TT and CT are 99.3% and 99.6% correspondingly. | PI*S variant of the SERPINA1 gene | | | | | |-----------------------------------|------------------------|---------------------------------|----|----| | | | Sanger Bidirectional Sequencing | | | | | Genotype | TT | AT | AA | | 23andMe | TT (homozygous common) | 65 | 0 | 0 | | PGS Test | AT (heterozygous) | 0 | 68 | 0 | | | AA (homozygous rare) | 0 | 0 | 69 | {24}------------------------------------------------ | 'no calls' or 'invalid" | 0 | 0 | 0 | |-------------------------|----|----|----| | Total | 65 | 68 | 69 | #### Percent Agreement (PA) of device results with comparator results of saliva samples: PA (TT|TT) = 100% (65/65) with 95% CI: 94.4% to 100% PA (AT|AT) = 100% (68/68) with 95% CI: 94.7% to 100% PA (AA|AA) =100% (69/69) with 95% CI: 94.7% to 100% Percent of no calls or invalid is 0.0% (0/202) with 95% CI: 0.0% to 1.9% Minor variant frequency for PI*S SERPINA1 in individuals of European descent in the 23andMe database was 7.97%; technical (analytical) positive predictive values for 23andMe PGS test result of TT and CT are 99.7% and 99.8% correspondingly. ### Comparison for Late-onset Alzheimer's Disease: The goal of the comparison study was to evaluate genotype assignment between the 23andMe PGS Test and a gold standard, Sanger bidirectional sequencing for the s4 variant of the APOE gene. The study design and acceptance criteria were identical to Hereditary Thrombophilia. | ε4 variant of the APOE gene | | | | | |-----------------------------|-------------------------|---------------------------------|-----|-----| | | | Sanger Bidirectional Sequencing | | | | | Genotype | TT | CT | CC | | 23andMe<br>PGS Test | TT (homozygous common) | 316 | 0 | 0 | | | CT (heterozygous) | 0 | 126 | 0 | | | CC (homozygous rare) | 0 | 1 | 101 | | | 'no calls' or 'invalid' | 4 | 2 | 5 | | Total | | 320 | 129 | 106 | #### Percent Agreement (PA) of device results with comparator results of saliva samples: PA (TT|TT) = 100% (316/316) with 95% CI: 98.8% to 100% PA (CT|CT) = 99.2% (126/127) with 95% CI: 95.7% to 99.9% PA (CC|CC) =100% (101/101) with 95% CI: 96.3% to 100% Percent of no calls or invalid is 2.0% (11/555) with 95% CI: 1.1% to 3.4% APOE &4 genotype frequencies in individuals of European descent reported in published literature are 1.8% for CC genotype and 23.9% for CT genotype; technical (analytical) positive predictive values for 23andMe PGS test results of CC and CT are 90.5% and 99.9% correspondingly. ### Comparison for Parkinson's Disease The goal of the comparison studies was to evaluate genotype assignment between the 23andMe PGS Test and a gold standard. Sanger bidirectional sequencing for the G2019S variant of the LRRK2 gene and the N370S variant of the GBA gene. The {25}------------------------------------------------ |…
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