23ANDME PERSONAL GENOME SERVICE (HEREINAFTER KNOWN AS PGS)

DEN140044 · 23AndMe · PKB · Feb 19, 2015 · Immunology

Device Facts

• Record ID: DEN140044
• Device Name: 23ANDME PERSONAL GENOME SERVICE (HEREINAFTER KNOWN AS PGS)
• Applicant: 23AndMe
• Product Code: PKB · Immunology
• Decision Date: Feb 19, 2015
• Decision: DENG
• Submission Type: Direct
• Regulation: 21 CFR 866.5940
• Device Class: Class 2

Intended Use

The 23andMe PGS Carrier Screening Test for Bloom Syndrome is indicated for the detection of the BLM^Ash variant in the BLM gene from saliva collected using an FDA cleared collection device (Oragene DX model OGD-500.001). This test can be used to determine carrier status for Bloom syndrome, but cannot determine if a person has two copies of the BLM^Ash variant. The test is most relevant for people of Ashkenazi Jewish descent.

Device Story

The 23andMe PGS Carrier Screening Test is a non-invasive, over-the-counter service for detecting the BLMAsh variant in the BLM gene. Users collect saliva in an Oragene-Dx kit, which stabilizes DNA for transport to a laboratory. The lab extracts DNA and processes it using a custom Illumina Infinium BeadChip. The Illumina iScan system uses laser-based optical imaging to excite fluorophores on the BeadChip; GenomeStudio and Coregen software analyze the resulting intensity data to determine genotypes. Results are reported to the user via a secure online account. The test helps adults of reproductive age determine carrier status for Bloom syndrome, facilitating informed reproductive decision-making. It does not diagnose disease or assess fetal health; users are advised to consult healthcare providers for result interpretation.

Clinical Evidence

No clinical data provided in the document. The order mandates that future performance validation must include accuracy studies (PPA/NPA > 99%) compared to bidirectional sequencing, precision/reproducibility studies, analytical specificity/sensitivity, and user comprehension studies (minimum 90% comprehension rate) for OTC labeling.

Technological Characteristics

Uses Illumina Infinium BeadChip (v4 assay, ~750,000 SNPs) for genotyping. Employs Tecan Evo for liquid handling and Illumina iScan for optical scanning. Software includes iScan Control, GenomeStudio (analysis), and Coregen (reporting). Saliva collected in Oragene-Dx OGD-500.001. Requires 15-50 ng/uL DNA concentration. Analysis is based on SNP-specific primer hybridization and hapten-labeled nucleotide extension.

Indications for Use

Indicated for autosomal recessive carrier screening for the BLMAsh variant in the BLM gene in adults of reproductive age, particularly those of Ashkenazi Jewish descent. Not for disease diagnosis, prenatal testing, risk assessment, prognosis, or newborn screening.

Regulatory Classification

Identification

Autosomal recessive carrier screening gene mutation detection system is a qualitative in vitro molecular diagnostic system used for genotyping of clinically relevant variants in genomic DNA isolated from human specimens intended for prescription use or over-the-counter use. The device is intended for autosomal recessive disease carrier screening in adults of reproductive age. The device is not intended for copy number variation, cytogenetic, or biochemical testing.

Special Controls

*Classification.* Class II (special controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 866.9, except § 866.9(c)(2). Autosomal recessive carrier screening gene mutation detection system must comply with the following special controls:(1) If the device is offered over-the-counter, the device manufacturer must provide information to a potential purchaser or actual test report recipient about how to obtain access to a board-certified clinical molecular geneticist or equivalent to assist in pre- and post-test counseling. (2) The device must use a collection device that is FDA cleared, approved, or classified as 510(k) exempt, with an indication for in vitro diagnostic use in DNA testing. (3) The device's labeling must include a prominent hyperlink to the manufacturer's public Web site where the manufacturer shall make the information identified in 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 prominently placed 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 prominently placed and 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) A detailed device description including: (A) Gene (or list of the genes if more than one) and variants the test detects (using standardized nomenclature, Human Genome Organization (HUGO) nomenclature, and coordinates). (B) Scientifically established clinical validity of each variant detected and reported by the test, which must be well-established in peer-reviewed journal articles, authoritative summaries of the literature such as Genetics Home Reference ( *http://ghr.nlm.nih.gov/* ), GeneReviews (*http://www.ncbi.nlm.nih.gov/books/NBK1116/* ), or similar summaries of valid scientific evidence, and/or professional society recommendations, including:( *1* ) Genotype-phenotype information for the reported mutations.( *2* ) Relevant American College of Medical Genetics (ACMG) or American Congress of Obstetricians and Gynecologists (ACOG) guideline recommending testing of the specific gene(s) and variants the test detects and recommended populations, if available. If not available, a statement stating that professional guidelines currently do not recommend testing for this specific gene(s) and variants.( *3* ) Table of expected prevalence of carrier status in major ethnic and racial populations and the general population.(C) The specimen type ( *e.g.,* saliva, whole blood), matrix, and volume.(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 ( *e.g.,* failed quality control) based on data from clinical samples, description of scenarios in which a test can fail (*i.e.,* low sample volume, low DNA concentration, etc.), how customers will be notified, and followup actions to be taken.(I) Specification of the criteria for test result interpretation and reporting. (ii) Information that demonstrates the performance characteristics of the device, including: (A) Accuracy (method comparison) of study results for each claimed specimen type. ( *1* ) Accuracy of the device shall be evaluated with fresh clinical specimens collected and processed in a manner consistent with the device'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 device's instructions for use, stored appropriately, and randomly selected. In some instances, use of contrived samples or human cell line samples may also be appropriate; the contrived or human cell line samples shall mimic clinical specimens as much as is feasible and provide an unbiased evaluation of the device's accuracy.( *2* ) Accuracy must be evaluated as compared to bidirectional sequencing or other methods identified as appropriate by FDA. Performance criteria for both the comparator method and device must be predefined and appropriate to the test's intended use. Detailed appropriate study protocols must be provided.( *3* ) Information provided shall include the number and type of specimens, broken down by clinically relevant variants, that were compared to bidirectional sequencing or other methods identified as appropriate by FDA. The accuracy, defined as positive percent agreement (PPA) and negative percent agreement (NPA), must be measured; accuracy point estimates must be greater than 99 percent (both per reported variant and overall) and uncertainty of the point estimate must be presented using the 95 percent confidence interval. Clinical specimens must include both homozygous wild type and heterozygous genotypes. The number of clinical specimens for each variant reported that must be included in the accuracy study must be based on the variant prevalence. Common variants (greater than 0.1 percent allele frequency in ethnically relevant population) must have at least 20 unique heterozygous clinical specimens tested. Rare variants (less than or equal to 0.1 percent allele frequency in ethnically relevant population) shall have at least three unique mutant heterozygous specimens tested. Any no calls (*i.e.,* absence of a result) or invalid calls (*e.g.,* failed quality control) in the study must be included in accuracy study results and reported separately. Variants that have a point estimate for PPA or NPA of less than 99 percent (incorrect test results as compared to bidirectional sequencing or other methods identified as appropriate by FDA) must not be incorporated into test claims and reports. Accuracy measures generated from clinical specimens versus contrived samples or cell lines must be presented separately. Results must be summarized and presented in tabular format, by sample and by genotype. Point estimate of PPA should be calculated as the number of positive results divided by the number of specimens known to harbor variants (mutations) without “no calls” or invalid calls. The point estimate of NPA should be calculated as the number of negative results divided by the number of wild type specimens tested without “no calls” or invalid calls, for each variant that is being reported. Point estimates should be calculated along with 95 percent two-sided confidence intervals.( *4* ) Information shall be reported on the clinical positive predictive value (PPV) and negative predictive value (NPV) for carrier status (and where possible, for each variant) in each population. Specifically, to calculate PPV and NPV, estimate test coverage (TC) and the percent of persons with variant(s) included in the device among all carriers: PPV = (PPA * TC * π)/(PPA * TC * π + (1 − NPA) * (1 − π)) and NPV = (NPA * (1 − π))/(NPA *(1 − π) + (1 − PPA*TC) * π) where PPA and NPA described either in paragraph (b)(3)(ii)(A)(*4* )(*i* ) or in paragraph (b)(3)(ii)(A)(*4* )(*ii* ) of this section and π is prevalence of carriers in the population (pre-test risk to be a carrier for the disease).( *i* ) For the point estimates of PPA and NPA less than 100 percent, use the calculated estimates in the PPV and NPV calculations.( *ii* ) Point estimates of 100 percent may have high uncertainty. If these variants are measured using highly multiplexed technology, calculate the random error rate for the overall device and incorporate that rate in the estimation of the PPA and NPA as calculated previously. Then use these calculated estimates in the PPV and NPV calculations. This type of accuracy study is helpful in determining that there is no systematic error in such devices.(B) Precision (reproducibility): Precision data must be generated using multiple instruments and multiple operators, on multiple non-consecutive days, and using multiple reagent lots. The sample panel must include specimens with claimed sample type ( *e.g.* saliva samples) representing different genotypes (*i.e.,* wild type, heterozygous). Performance criteria must be predefined. A detailed study protocol must be created in advance of the study and then followed. The “failed quality control” rate must be indicated. It must be clearly documented whether results were generated from clinical specimens, contrived samples, or cell lines. The study results shall state, in a tabular format, the variants tested in the study and the number of replicates for each variant, and what testing conditions were studied (*i.e.,* number of runs, days, instruments, reagent lots, operators, specimens/type, etc). The study must include all nucleic acid extraction steps from the claimed specimen type or matrix, unless a separate extraction study for the claimed sample type is performed. If the device is to be used at more than one laboratory, different laboratories must be included in the precision study (and reproducibility must be evaluated). The percentage of “no calls” or invalid calls, if any, in the study must be provided as a part of the precision (reproducibility) study results.(C) Analytical specificity data: Data must be generated evaluating the effect on test performance of potential endogenous and exogenous interfering substances relevant to the specimen type, evaluation of cross-reactivity of known cross-reactive alleles and pseudogenes, and assessment of cross-contamination. (D) Analytical sensitivity data: Data must be generated demonstrating the minimum amount of DNA that will enable the test to perform accurately in 95 percent of runs. (E) Device stability data: The manufacturer must establish upper and lower limits of input nucleic acid and sample stability that will achieve the claimed accuracy and reproducibility. Data supporting such claims must be described. (F) Specimen type and matrix comparison data: Specimen type and matrix comparison data must be generated if more than one specimen type or anticoagulant can be tested with the device, including failure rates for the different specimen types. (iii) If the device is offered over-the-counter, including cases in which the test results are provided direct-to-consumer, the manufacturer must conduct a study that assesses user comprehension of the device's labeling and test process and provide a concise summary of the results of the study. The following items must be included in the user study: (A) The test manufacturer must perform pre- and post-test user comprehension studies to assess user ability to understand the possible results of a carrier test and their clinical meaning. The comprehension test questions must directly evaluate the material being presented to the user in the test reports. (B) The test manufacturer must provide a carrier testing education module to potential and actual test report recipients. The module must define terms that are used in the test reports and explain the significance of carrier status. (C) The user study must meet the following criteria: ( *1* ) The study participants must be comprised of a statistically justified and demographically diverse population (determined using methods such as quota-based sampling) that is representative of the intended user population. Furthermore, the users must be comprised of a diverse range of age and educational levels that have no prior experience with the test or its manufacturer. These factors shall be well-defined in the inclusion and exclusion criteria.( *2* ) All sources of bias (*e.g.,* non-responders) must be predefined and accounted for in the study results with regard to both responders and non-responders.( *3* ) The testing must follow a format where users have limited time to complete the studies (such as an 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).( *4* ) Users must be randomly assigned to study arms. Test reports given to users must: Define the condition being tested and related symptoms; explain the intended use and limitations of the test; explain the relevant ethnicities regarding the variant tested; explain carrier status and relevance to the user's ethnicity; and provide links to additional information pertaining to situations where the user is concerned about their test results or would like followup information as indicated in test labeling. The study shall assess participants' ability to understand the following comprehension concepts: The test's limitations, purpose, and results.( *5* ) Study participants must be untrained, naive to the test subject of the study, and be provided only the materials that will be available to them when the test is marketed.( *6* ) The user comprehension study must meet the predefined primary endpoint criteria, including a minimum of a 90 percent or greater overall comprehension rate (*i.e.* selection of the correct answer) for each comprehension concept to demonstrate that the education module and test reports are adequate for over-the-counter use.(D) A summary of the user comprehension study must be provided and include the following: ( *1* ) Results regarding reports that are provided for each gene/variant/ethnicity tested.( *2* ) Statistical methods used to analyze all data sets.( *3* ) Completion rate, non-responder rate, and reasons for non-response/data exclusion, as well as a summary table of comprehension rates regarding comprehension concepts (purpose of test, test results, test limitations, ethnicity relevance for the test results, etc.) for each study report.(4) Your 21 CFR 809.10 compliant labeling and any test report generated must include the following warning and limitation statements, as applicable: (i) A warning that reads “The test is intended only for autosomal recessive carrier screening in adults of reproductive age.” (ii) A statement accurately disclosing the genetic coverage of the test in lay terms, including, as applicable, information on variants not queried by the test, and the proportion of incident disease that is not related to the gene(s) tested. For example, where applicable, the statement would have to include a warning that the test does not or may not detect all genetic variants related to the genetic disease, and that the absence of a variant tested does not rule out the presence of other genetic variants that may be disease-related. Or, where applicable, the statement would have to include a warning that the basis for the disease for which the genetic carrier status is being tested is unknown or believed to be non-heritable in a substantial number of people who have the disease, and that a negative test result cannot rule out the possibility that any offspring may be affected with the disease. The statement would have to include any other warnings needed to accurately convey to consumers the degree to which the test is informative for carrier status. (iii) For prescription use tests, the following warnings that read: (A) “The results of this test are intended to be interpreted by a board-certified clinical molecular geneticist or equivalent and should be used in conjunction with other available laboratory and clinical information.” (B) “This device is not intended for disease diagnosis, prenatal testing of fetuses, risk assessment, prognosis or pre-symptomatic testing, susceptibility testing, or newborn screening.” (iv) For over-the-counter tests, a statement that reads “This test is not intended to diagnose a disease, or tell you anything about your risk for developing a disease in the future. On its own, this test is also not intended to tell you anything about the health of your fetus, or your newborn child's risk of developing a particular disease later on in life.” (v) For over-the-counter tests, the following warnings that read: (A) “This test is not a substitute for visits to a healthcare provider. It is recommended that you consult with a healthcare provider if you have any questions or concerns about your results.” (B) “The test does not diagnose any health conditions. Results should be used along with other clinical information for any medical purposes.” (C) “The laboratory may not be able to process your sample. The probability that the laboratory cannot process your saliva sample can be up to [actual probability percentage].” (D) “Your ethnicity may affect how your genetic health results are interpreted.” (vi) For a positive result in an over-the-counter test when the positive predictive value for a specific population is less than 50 percent and more than 5 percent, a warning that reads “The positive result you obtained may falsely identify you as a carrier. Consider genetic counseling and followup testing.” (vii) For a positive result in an over-the-counter test when the positive predictive value for a specific population is less than 5 percent, a warning that reads “The positive result you obtained is very likely to be incorrect due to the rarity of this variant. Consider genetic counseling and followup testing.” (5) The testing done to comply with paragraph (b)(3) of this section must show the device meets or exceeds each of the following performance specifications: (i) The accuracy must be shown to be equal to or greater than 99 percent for both PPA and NPA. Variants that have a point estimate for PPA or NPA of less than 99 percent (incorrect test results as compared to bidirectional sequencing or other methods identified as appropriate by FDA) must not be incorporated into test claims and reports. (ii) Precision (reproducibility) performance must meet or exceed 99 percent for both positive and negative results. (iii) The user comprehension study must obtain values of 90 percent or greater user comprehension for each comprehension concept. (6) The distribution of this device, excluding the collection device described in paragraph (b)(2) of this section, shall be limited to the manufacturer, the manufacturer's subsidiaries, and laboratories regulated under the Clinical Laboratory Improvement Amendments.

Submission Summary (Full Text)

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#### EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR The 23andMe Personal Genome Service Carrier Screening Test for Bloom Syndrome

#### DECISION SUMMARY

This decision summary corrects the decision summary dated February 2015.

#### A. DEN Number:

DEN140044

#### B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the 23andMe Personal Genome Service (PGS) Carrier Screening Test for Bloom Syndrome

#### C. Measurands:

Genomic DNA obtained from a human saliva sample

#### D. Type of Test:

The 23andMe PGS Carrier Screening Test for Bloom Syndrome, using the Illumina Infinium BeadChip (23andMe BeadChip), is designed to be capable of detecting specific single nucleotide polymorphisms (SNPs) as well as other genetic variants. The 23andMe PGS Carrier Screening Test for Bloom Syndrome is a molecular assay indicated for use for the detection of the BLMAsh variant in the BLM gene from saliva collected using the OrageneDx® saliva collection device (OGD-500.001). Results are analyzed using the Illumina iScan System and Genome Studio and Coregen software. The 23andMe PGS Carrier Screening Test for Bloom Syndrome can be used to determine carrier status for Bloom syndrome, but cannot determine if a person has two copies of the BLMAS variant.

## E. Applicant:

23andMe, Inc.

#### F. Proprietary and Established Names:

23andMe Personal Genome Service Carrier Screening Test for Bloom Syndrome

## G. Regulatory Information:

- 1. Regulation section:
- 21 CFR 866.5940
- 2. Classification:

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Class II

- 3. Product code(s):
PKB

- 4. Panel:
82- Immunology

# H. Indication(s) for use:

- 1. Indication(s) for use:
The 23andMe PGS Carrier Screening Test for Bloom Syndrome is indicated for the detection of the BLMAsh variant in the BLM gene from saliva collected using an FDA cleared collection device (Oragene DX model OGD-500.001). This test can be used to determine carrier status for Bloom syndrome in adults of reproductive age, but cannot determine if a person has two copies of the BLM140 variant. The test is most relevant for people of Ashkenazi Jewish descent.

# 2. Special conditions for use statement(s):

1. For over-the-counter (OTC) use.

2. This test is not intended to diagnose a disease, to tell you anything about the health of your fetus, or your risk or your new born child's risk of developing a particular disease later on in life.

3. This test is not a substitute for visits to a healthcare provider. It is recommended that you consult with a healthcare provider if you have any questions or concerns about your results.

4. 23andMe PGS Carrier Screening Test for Bloom Syndrome does not detect all genetic variants associated with Bloom Syndrome. The absence of a variant tested does not rule out the presence of other genetic variants that may be disease related.

5. The test is intended only for autosomal recessive carrier screening in adults of reproductive age.

6. The test does not diagnose any health conditions. Results should be used along with other clinical information for any medical purposes.

7. The laboratory may not be able to process a patient's sample. The probability that the laboratory cannot process the sample can be up to 7.6%.

8. A user's ethnicity may affect how the genetic test results are interpreted.

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- 9. Subject to meeting the limitations contained in the special controls under regulation 21 CFR 866.5940.
#### 4. Special instrument requirements:

The 23andMe PGS Carrier Screening Test for Bloom Syndrome is 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 analytical genotype profile for each sample. The data is used to generate test reports that are based on information from reported scientific findings on genotypes.

## I. Device Description:

The 23andMe Personal Genome Service (PGS) Carrier Screening Test for Bloom Syndrome (hereafter the "PGS") is a non-invasive genetic information service that combines qualitative genotyping data for an individual. The PGS is indicated for use for the detection of the BLM4sh variant in the BLM gene from saliva collected using the Oragene•Dx Saliva Collection Device (Oragene Dx model OGD-500.01). The core components of the PGS consist of the saliva collection kit: custom genotyping chip: laboratory procedures, equipment and analysis; and result reporting software.

The saliva collection kit includes a sample collection tube with a unique barcode printed by the manufacturer, funnel, preservative solution, instructions for use, and pre-paid packaging for returning the sample to the processing laboratory. Saliva may be collected by spitting directly into the Oragene Dx container or may be transferred into the Oragene Dx container using a sponge. Saliva samples collected using Oragene•Dx are stabilized and can be transported and/or stored long term at ambient conditions.

The PGS is indicated for the detection of the BLMAsh variant in the BLM gene using DNA extracted from 2 mL saliva samples that are collected in a FDA cleared or approved collection device. Illumina manufactures a custom Infinium BeadChip genotyping chip for the device. The chip is designed to detect specific single nucleotide polymorphisms (SNPs) as well as other genetic variants; all markers refer to specific positions in the National Center for Biotechnology Information (NCBI) reference human genome.

After placing an order, an individual receives via post an Oragene-Dx saliva collection kit. Once the saliva sample is received by the laboratory. DNA extraction and quantitation steps occur. Samples meeting a minimum DNA concentration of 15 ng/uL are processed and prepared for amplification and BeadChip addition. BeadChips are read by the Illumina iScan, which is a laser-based, high-resolution optical imaging system. The instrument reads BeadChips by employing red and green lasers to excite the fluorophores of the allele-specific extended products found on the beads. Light emissions from these fluorophores are then recorded in high-resolution images of each BeadChip section. Data from these are analyzed to determine genotypes using Illumina's GenomeStudio software package. GenomeStudio is a modular software application that allows viewing and analyzing of

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genotypic data obtained from the iScan.

## J. Substantial Equivalence Information:

- 1. Predicate device name(s):
No predicate device exists.

- 2. Predicate 510(k) number(s):
Not applicable.

- 3. Comparison with predicate:
Not applicable.

#### K. Standard/Guidance Document Referenced (if applicable):

Not applicable.

#### L. Test Principle:

The PGS is indicated to be performed using the BeadChip v4 assay (Illumina HumanOmniExpress-24 format chip), which covers ~750,000 SNPs. 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 SNP-specific primers, followed by extension with hapten-labeled nucleotides. The primers hybridize adjacent to the SNPs and are extended with a single nucleotide corresponding to the SNP 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/processing and BeadChip quantification and scanning, respectively. The genotype content is separated, analyzed, and then integrated into pre-defined report templates specific for each condition associated with each genotype. The iScan software uses the dmap file to associate signal intensity measured by the iScan Reader with bead type. The algorithm uses sequential hybridizations of dye-labeled oligonucleotides, or decoders, complementary to bead sequences to create a combinatorial decoding scheme for arravs. The approach uses longer sequences; each is designed to hybridize to a defined target with high specificity. It is capable of decoding, with high accuracy, many thousands of bead types. Each bead type is defined by a unique DNA sequence that is recognized by a complementary decoder. Genotypes are determined using the GenomeStudio software package.

The PGS is indicated to detect the BLMAsh variant. The BLMAsh variant results from a deletion of six letters and an insertion of seven letters in the sequence of the gene. It results in a shortened protein that does not work properly (Typical Sequence → Deletion and Insertion > TAGATTC Variant Sequence).

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Intensity values that fall outside of the expected range of intensities are returned as "no-calls"; however, all homozygous variant (BLMAsh II) genotype samples receive a "no call" result, since the calling software was designed not to detect BLMdsb II homozygous variant genotypes. Such results will be reported as "could not be determined" to the customer in their test report. Genetic results are returned to the customer in a secure online account on the 23andMe website.

## 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:

#### a. Precision/Reproducibility

For evaluation of precision/reproducibility, two studies were conducted: the first study was conducted with human cell line samples and a second study was conducted with human saliva samples.

Precision Study with Human Cell Line Samples

The precision study was conducted at the b(4) TS/CCI Six DNA samples taken from b(4) TS/CCI cell b(4) TS/CCI lines (4 BLM homozygous common ("DD") samples, 1 BLMAs" heterozygous ("DI") sample and 1 BLMAsh homozygous rare ("II") sample) were tested over 5 days by 3 operator teams at site 1 and 33 operator teams at site 2. To confirm the BeadChip genotype, each [4] TS/CC sample was also sequenced by bi-directional Sanger Sequencing. Samples were tested with 4 lots of reagents (lot 1, lot 2 and lot 3 at site 1 and lot 2, lot 3 and lot 4 at site 2) using 3 Tecan instruments and 3 iScan instruments in different combinations. For each of 4 "DD" samples, there were 36 replicates per day per laboratory site with total number of 360 replicates (36 x 5 x 2). The other two samples, "DI" and "II", had 72 replicates per day per site with total number of 720 replicates (72 x 5 x 2). Information regarding samples that failed quality control (FQC) was also evaluated.

Results of the Study Stratified by Site are presented below:

| Site 1 | Total number<br>of replicates | Number of<br>correct calls | Number of<br>miscalls | Number of<br>"FQC"<br>replicates | Percent of<br>"FQC"<br>replicates |
|------------------|-------------------------------|----------------------------|-----------------------|----------------------------------|-----------------------------------|
| Sample 1<br>"DD" | 180 | 173 | 0 | 7 | 3.89% |
| Sample 2<br>"DD" | 180 | 174 | 0 | 6 | 3.33% |
| Sample 3 | 180 | 175 | 0 | 5 | 2.78% |

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| "DD" | | | | | |
|------------------|-------|-------|---|----|-------|
| Sample 4<br>"DD" | 180 | 175 | 0 | 5 | 2.78% |
| Sample "DI" | 360 | 350 | 0 | 10 | 2.78% |
| Sample "II" | 360 | 343 | 0 | 17 | 4.72% |
| Total | 1,440 | 1,390 | 0 | 50 | 3.47% |

| Site 2 | Total number<br>of replicates | Number of<br>correct calls | Number of<br>miscalls | Number of<br>"FQC"<br>replicates | Percent of<br>"FQC"<br>replicates |
|------------------|-------------------------------|----------------------------|-----------------------|----------------------------------|-----------------------------------|
| Sample 1<br>"DD" | 180 | 171 | 0 | 9 | 5.00% |
| Sample 2<br>"DD" | 180 | 174 | 0 | 6 | 3.33% |
| Sample 3<br>"DD" | 180 | 176 | 0 | 4 | 2.22% |
| Sample 4<br>"DD" | 180 | 179 | 0 | 1 | 0.56% |
| Sample "DI" | 360 | 350 | 0 | 10 | 2.78% |
| Sample "II" | 360 | 350 | 0 | 10 | 2.78% |
| Total | 1,440 | 1,400 | 0 | 40 | 2.78% |

Results of the study (percent of "FQC" replicates) stratified by instrument combinations are shown for each site below:

Percent of "FQC" for nine different combinations of 3 Tecan and 3 iScan instruments at Percent
Site

| | | Percent of "FQC" | | | |
|-------|-------|-------------------|------------------|------------------|-------|
| Tecan | iScan | 4 Samples<br>"DD" | Sample "DI" | Sample "II" | Total |
| 1 | 1 | 0.00%<br>(0/96) | 0.00%<br>(0/48) | 0.00%<br>(0/48) | 0.00% |
| 1 | 2 | 0.00%<br>(0/48) | 4.17%<br>(1/24) | 4.17%<br>(1/24) | 2.08% |
| 1 | 3 | 8.33%<br>(8/96) | 4.17%<br>(2/48) | 2.08%<br>(1/48) | 5.73% |
| 2 | 1 | 0.00%<br>(0/96) | 0.00%<br>(0/48) | 0.00%<br>(0/48) | 0.00% |
| 2 | 2 | 7.29%<br>(7/96) | 4.17%<br>(2/48) | 14.58%<br>(7/48) | 8.33% |
| 2 | 3 | 4.17%<br>(2/48) | 12.50%<br>(3/24) | 12.50%<br>(3/24) | 8.33% |
| 3 | 1 | 4.17%<br>(2/48) | 0.00%<br>(0/24) | 0.00%<br>(0/24) | 2.08% |
| 3 | 2 | 0.00%<br>(0/48) | 0.00%<br>(0/48) | 0.00%<br>(0/48) | 0.00% |

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| 3 | 3 | 4.17% | 4.17% | 10.42% | | | | |
|-------------------------------|-------|-------------------|-------------|-------------|-------|--|--|--|
| | | (4/96) | (2/48) | (5/48) | 5.73% | | | |
| Percent of | | | | | | | | |
| "FQC" for | | | | | | | | |
| nine different | | | | | | | | |
| combinations | | | | | | | | |
| of 3 Tecan | | Percent of "FQC" | | | | | | |
| and 3 iScan | | | | | | | | |
| instruments at<br>b(4) TS/CCl | | | | | | | | |
| Tecan | iScan | 4 Samples<br>"DD" | Sample "DI" | Sample "II" | Total | | | |
| 4 | 4 | 4.17% | 0.00% | 4.17% | 3.13% | | | |
| | | (4/96) | (0/48) | (2/48) | | | | |
| 4 | 5 | 2.08% | 2.08% | 6.25% | 3.13% | | | |
| | | (2/96) | (1/48) | (3/48) | | | | |
| 4 | 6 | 0.00% | 0.00% | 0.00% | 0.00% | | | |
| | | (0/48) | (0/24) | (0/24) | | | | |
| 5 | 4 | 0.00% | 5.36% | 0.00% | 1.34% | | | |
| | | (0/112) | (3/56) | (0/56) | | | | |
| 5 | 5 | 6.25% | 0.00% | 0.00% | 3.13% | | | |
| | | (3/48) | (0/24) | (0/24) | | | | |
| 5 | 6 | 12.5% | 7.50% | 2.50% | 8.75% | | | |
| | | (10/80) | (3/40) | (1/40) | | | | |
| 6 | 4 | 0.00% | 0.00% | 0.00% | 0.00% | | | |
| | | (0/48) | (0/24) | (0/24) | | | | |
| 6 | 5 | 1.04% | 4.17% | 4.17% | 2.60% | | | |
| | | (1/96) | (2/48) | (2/48) | | | | |
| 6 | 6 | 0.00% | 2.08% | 4.17% | 1.56% | | | |
| | | (0/96) | (1/48) | (2/48) | | | | |

The percent of FQC for 9 combinations of Tecan and iScan instruments at "frises ranged from 0% to 8.33%. The percent of FQC for 9 combinations of Tecan and
iScan instruments at 7(4) TSCCI ranged from 0% to 8.75%

Results of the Study (percent of "FQC" replicates) Stratified by Lot of Reagents are presented below:

| | Sample 1<br>"DD" | Sample 2<br>"DD" | Sample 3<br>"DD" | Sample 4<br>"DD" | Sample<br>"DI" | Sample<br>"II" | Total |
|-------|------------------|------------------|-------------------|------------------|------------------|-------------------|--------------------------|
| Lot 1 | 8.33%<br>(5/60) | 8.33%<br>(5/60) | 0.00%<br>(0/60) | 0.00%<br>(0/60) | 5.00%<br>(6/120) | 8.33%<br>(10/120) | <b>5.42%</b><br>(26/480) |
| Lot 2 | 5.00%<br>(6/120) | 0.83%<br>(1/120) | 2.50%<br>(3/1200) | 0.00%<br>(0/120) | 1.67%<br>(4/240) | 3.75%<br>(9/240) | <b>2.40%</b><br>(23/960) |
| Lot 3 | 4.17%<br>(5/120) | 4.17%<br>(5/120) | 5.00%<br>(6/120) | 4.17%<br>(5/120) | 1.67%<br>(4/240) | 1.67%<br>(4/240) | <b>3.02%</b><br>(29/960) |

{7}------------------------------------------------

| Lot 4 | 0.00% | 1.67% | 0.00% | 1.67% | 5.00% | 3.33% | <b>2.50%</b> |
|-------|--------|--------|--------|--------|---------|---------|--------------|
| | (0/60) | (1/60) | (0/60) | (1/60) | (6/120) | (4/120) | (12/480) |

The percent of "FQC" replicates for 4 different lots of reagents ranged from 2.4% to 5.4%.

The combined data of the reproducibility study for 6 human cell line samples are presented in the table below:

| | Total number<br>of replicates | Number of<br>correct calls | Number of<br>miscalls | Number of<br>"FQC"<br>replicates | Percent of<br>"FQC"<br>replicates |
|------------------|-------------------------------|----------------------------|-----------------------|----------------------------------|-----------------------------------|
| Sample 1<br>"DD" | 360 | 344 | 0 | 16 | 4.44% |
| Sample 2<br>"DD" | 360 | 348 | 0 | 12 | 3.33% |
| Sample 3<br>"DD" | 360 | 351 | 0 | 9 | 2.50% |
| Sample 4<br>"DD" | 360 | 354 | 0 | 6 | 1.67% |
| Sample "DI" | 720 | 700 | 0 | 20 | 2.78% |
| Sample “II” | 720 | 693 | 0 | 27 | 3.75% |
| Total | 2,880 | 2,790 | 0 | 90 | 3.13% |

96.9% (2,790/2,880) replicates produced correct genotyping results and 3.1% (90/2,880) replicates did not pass Quality Control (QC) acceptance criteria. Samples with failed QC on the first run are re-tested per laboratory SOPs; therefore, an anticipated rate of samples with two times failed QC based on precision study data of the human cell line samples is 0.1% (=0.0313 x 0.0313).

#### Laboratory Reproducibility Study with Saliva Samples

A reproducibility study was performed at the same 2 sites as the reproducibility study with human cell line samples with a total of 105 BLMAsh homozygous common ("DD") saliva samples obtained from individuals using the 23andMe Saliva Collection kit (Oragene-DX, OGD500.001, saliva collection kit). Sample processing was performed at both sites and tested with PGS test for Bloom syndrome. Fifty samples were initially processed at the first site and 55 samples were processed at the second site. A sample swap was performed, where an aliquot of the initially processed samples were shipped to the other lab. Results for 105 saliva samples are presented below for both sites.

{8}------------------------------------------------

| | | Saliva samples “DD” | |
|----------------------------------------------------|------------------------------------|---------------------|--------|
| | | Site 1 | Site 2 |
| PGS<br>Carrier<br>test<br>for<br>Bloom<br>Syndrome | First run<br>"1 Variant Detected" | 0 | 0 |
| | First run<br>"0 Variants Detected" | 104 | 87 |
| First<br>run<br>FQC<br>Result | Re-run<br>"1 Variant Detected" | 0 | 0 |
| | Re-run<br>"0 Variants Detected" | 1 | 10 |
| | Re-run<br>FQC | 0 | 8 |
| Total | | 105 | 105 |

Laboratory Reproducibility Study with First Run and Re-run Summary Results

Since the only samples tested were homozygous common (DD), there were no results for the "1 Variant Detected" category for either site. The percent of saliva samples with "FOC" on the first run was 1.0% (1/105) at site 1 and 17.1% (18/105) at site 2. Samples with the "FQC" on the first run were re-tested and the percent of saliva samples with a final failed QC result (Re-run FQC) was 0% (0/105) at site 1 and 7.6% (8/105) at site 2.

- b. Linearity/assay Reportable Range:
Not applicable

- c. Traceability, Stability, Expected Values (controls, calibrators, or methods):
The PGS requires two types of controls, the sample processing control and the reproducibility control. The sample processing control material is generated from b(4) TS/CCl

the 23andMe BeadChip according to routine Standard Operating Procedures (SOPs) at the contracted laboratory sites.

The reproducibility control consists of b(4) TS/CCI

Each new lot of the

reproducibility control is tested by comparison with reference BeadChip genotype results. DNA is extracted from this cell suspension and genotyped using the 23andMe BeadChip, according to routine SOPs at the contracted laboratory.

{9}------------------------------------------------

The sample processing control is routinely run on every sample genotyping plate and the reproducibility control is routinely run approximately once per week. Historical data from all such runs were analyzed for one lot of the sample processing control spanning 3 months and one lot of the reproducibility control spanning 1 year.

Stability protocols and acceptance criteria were reviewed and deemed acceptable. The information provided demonstrates that the sample processing control is stable for up to 3 months and the reproducibility control is stable for up to 12 months.

- d. Detection Limit:
Limit of Detection testing was performed using DNA samples [4) Tscc cell lines. The following samples/genotypes/replicates were tested: BLMAsh (homozygous wild type, DD), 4 samples/4 replicates per sample; BLMAsh (heterozygous variant, DI), 1 sample/8 replicates per sample; BLMAsh (homozygous variant, II), 1 sample/8 replicates per sample. Each DNA sample was tested at the following concentrations: 5, 15, and 50 ng/μL. BeadChip genotyping was performed [4] Tscc] and b(4) TS/CCI DNA, where each site tested the same DNA sample replicates for each of 3 BLMAsh genotypes at 3 DNA concentrations, with 3 lots of reagents. To confirm the BeadChip genotype, each 64756Cl sample was also sequenced by bi-directional Sanger sequencing. BeadChip genotypes were compared with sequenced genotypes to determine the rates of correct BeadChip genotype calls at each DNA concentration. If a sample replicate failed BeadChip or sequencing Quality Control (OC) criteria, it was marked as "FQC" ("failed QC") if the sample replicate did not demonstrate a call rate ≥ 0.980.

The lower LoD was defined as the lowest DNA concentration at which at least 95% of samples yielded the correct call at each of two laboratory sites. The LoD study vielded 100% correct call rates for all samples across all reagent lots, at all sample concentrations tested at two independent laboratory sites. Therefore, the study passed the acceptance criteria of 95% correct calls at the lowest concentration tested (5 ng/uL). The performance requirement for the PGS, has been set at a minimum of 15 ng/uL DNA and maximum of 50 ng/uL DNA.

Results from the studies also indicated that all but one replicate passed OC acceptance criteria on the re-run (per laboratory SOP) and yielded correct genotype calls. One replicate failed QC (due to call rate < 0.980) on both runs at the b(4) TS/CC laboratory site, with one lot of reagent, at the 5 ng/uL concentration.

#### e. Interfering Substances

#### Endogenous Interference Study

A study was conducted to determine whether endogenous substances present during saliva collection affect BLMAsh genotyping results. Saliva samples were collected from 10 individuals (with the homozygous common "DD" genotype) and sent to the contracted laboratory for DNA extraction and BeadChip genotyping. Saliva samples were split b(4) TS/CCI for addition of interfering endogenous substances. The

{10}------------------------------------------------

following substances were spiked separately into an aliquot of each saliva sample before DNA extraction:

- · salivary a-amylase, 395 U/mL
- · hemoglobin, 20 mg/mL
- · immunoglobulin A (IgA) 0.43 mg/mL

· total protein 2.67 mg/mL (composed of 0.185 mg/mL salivary o-amylase, 0.43 mg/mL IgA, and 2.05 mg/mL Human Serum Albumin)

An additional saliva sample aliquot was not spiked and served as control. Three replicates were tested for each aliquot. For each replicate, an aliquot of the saliva sample was processed according to laboratory SOPs to extract DNA. Extracted DNA was diluted to a concentration within the analytical range. The minimum required DNA concentration was 15 ng/uL. All replicates were tested on the same day, with one lot of reagents, by one operator team, using one set of instruments.

The PGS genotypes of the samples containing each endogenous substance were compared with the genotype of the control sample to determine percent concordance. The acceptance criteria were defined as a minimum of 95% concordant genotype calls across all individual samples for each of the 4 interfering substance conditions.

| Sample | Interfering<br>Substance | Replicate<br>Genotypes | | | | Correct | Incorrect | No<br>Call | FQC | %<br>Concordance<br>with control |
|--------|--------------------------|------------------------|----------|----------|---|---------|-----------|------------|-----|----------------------------------|
| | | Rep<br>1 | Rep<br>2 | Rep<br>3 | | | | | | |
| 1 | Control | DD | DD | DD | | | 0 | 0 | | |
| | Amylase | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Hemoglobin | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | IgA | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Total<br>protein | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| 2 | Control | DD | DD | DD | | | 0 | 0 | | |
| | Amylase | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Hemoglobin | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | IgA | DD | FQ | FQC | 1 | 0 | 0 | 2 | 100 | |
| | Total<br>protein | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| 3 | Control | DD | DD | DD | | | 0 | 0 | | |
| | Amylase | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Hemoglobin | DD | DD | FQC | 2 | 0 | 0 | 1 | 100 | |
| | IgA | DD | DD | FQC | 2 | 0 | 0 | 1 | 100 | |
| | Total<br>protein | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Control | DD | DD | DD | | | 0 | 0 | | |
| 4 | Amylase | DD | FQ | DD | 2 | 0 | 0 | 1 | 100 | |
| | Hemoglobin | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | IgA | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Total<br>protein | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Control | DD | DD | DD | | | 0 | 0 | | |
| | Amylase | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| 5 | Hemoglobin | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | IgA | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Total<br>protein | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Control | DD | DD | DD | | | 0 | 0 | | |
| | Amylase | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| 7 | Hemoglobin | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | IgA | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Total<br>protein | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Control | DD | DD | DD | | | 0 | 0 | | |
| | Amylase | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| 8 | Hemoglobin | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | IgA | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Total<br>protein | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Control | DD | DD | DD | | | 0 | 0 | | |
| | Amylase | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| 9 | Hemoglobin | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | IgA | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Total<br>protein | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Control | DD | DD | DD | | | 0 | 0 | | |
| 10 | Amylase | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Hemoglobin | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | IgA | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Total<br>protein | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| 11 | Control | DD | DD | DD | | | 0 | 0 | | |
| | Amylase | DD | FQC | FQC | 1 | 0 | 0 | 2 | 100 | |
| | Hemoglobin | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | IgA | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |
| | Total<br>protein | DD | DD | DD | 3 | 0 | 0 | 0 | 100 | |

Results of the study are shown in the table below:

{11}------------------------------------------------

{12}------------------------------------------------

FQC = Replicate failed QC according to QC criteria; FQC replicates were re-run on a separate dav

Out of a total 150 replicates run, 7/150 (4.7%) failed OC in this study. Of these, 5 replicates did not meet the QC criterion "call rate ≥ 0.980". Another 2 replicates had insufficient saliva volume to perform the first run. All 5 replicates with sufficient saliva volume produced the correct genotype upon re-run. The results demonstrate that there was no negative impact upon PGS test performance with all interferents tested.

#### Exogenous Interference Study

A study was performed to determine whether exogenous substances present in saliva during saliva collection would affect successful assignment of the correct BLMAsh genotype using the 23andMe BeadChip assay.

Saliva samples were collected from 5 individuals at 3 time points - before consuming an exogenous substance (baseline), immediately (0 minutes) after, and 30 minutes after - and sent to the laboratory for genotyping. For each individual. genotype test results of the 0 and 30 minute samples were compared with genotypes of the baseline sample (considered control) to determine percent concordance. The following exogenous interference conditions were tested separately:

- Eating (food containing beef) .
- . Eating (food other than beef)
- . Drinking
- . Chewing gum
- . Using mouthwash

Each exogenous substance sample was tested in triplicate for a total of 225 test samples (5 donors x 5 conditions x 3 time points x 3 replicates). For each replicate, an aliquot of the saliva sample was processed according to laboratory SOPs to extract DNA. Extracted DNA was diluted to a concentration within the analytical range. The minimum required DNA concentration was 15 ng/uL. The acceptance criteria were defined as a minimum of 95% concordant genotype calls across all individual samples at each time point. The study results are shown below:

{13}------------------------------------------------

| Exogenous<br>substance<br>activity | Individual<br>ID | Time<br>point | Genotype | | | Concordance results | | | | |
|------------------------------------|------------------|---------------|----------|-------|-------|---------------------|-----------|-----------|---------|-----|
| | | | rep 1 | rep 2 | rep 3 | | Correct | Incorrect | No Call | FQC |
| | 1 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | | 30 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | 2 | baseline | DD | DD | DD | | | | | |
| | | 0 min | FQC | FQC | DD | | 1 | 0 | 0 | 2 |
| | | 30 min | FQC | DD | DD | | 2 | 0 | 0 | 1 |
| Eating<br>(beef) | 3 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | | 30 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | 4 | baseline | DD | FQC | DD | | | | | 1 |
| | | 0 min | FQC | FQC | FQC | | all FQC | 0 | 0 | 3 |
| | | 30 min | DD | FQC | DD | | 2 | 0 | 0 | 1 |
| | 5 | baseline | DD | DD | DD | | | | | |
| | | 0 min | FQC | FQC | FQC | | all FQC | 0 | 0 | 3 |
| | | 30 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | 1 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | FQC | | 2 | 0 | 0 | 1 |
| | | 30 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | 2 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | | 30 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| Eating<br>(no beef) | 3 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | | 30 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | 4 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | | 30 min | DD | DD | DD | | 3 | 0 | 0 | 0 |
| | 5 | baseline | DD | FQC | DD | | | | | 1 |
| | | 0 min | FQC | FQC | FQC | all FQC | 0 | 0 | 3 | |
| | | 30 min | FQC | DD | DD | 2 | 0 | 0 | 1 | |
| | 1 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | 2 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | 3 | baseline | DD | FQC | DD | | | | 1 | |
| Drinking | | 0 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | 4 | baseline | DD | FQC | DD | | | | 1 | |
| | | 0 min | DD | FQC | FQC | 1 | 0 | 0 | 2 | |
| | | 30 min | DD | FQC | FQC | 1 | 0 | 0 | 2 | |
| | 5 | baseline | DD | DD | DD | | | | | |
| | | 0 min | FQC | FQC | DD | 1 | 0 | 0 | 2 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| Exogenous<br>substance<br>activity | Individual<br>ID | Time<br>point | rep 1 | rep 2 | rep 3 | Correct | Incorrect | No Call | FQC | |
| | 1 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | 2 | baseline | DD | DD | DD | | | | | |
| Chewing<br>gum | | 0 min | DD | FQC | FQC | 1 | 0 | 0 | 2 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | 3 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | | baseline | DD | DD | DD | | | | | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | 5 | baseline | DD | DD | DD | | | | | |
| | | 0 min | FQC | DD | DD | 2 | 0 | 0 | 1 | |
| | | 30 min | FQC | DD | DD | 2 | 0 | 0 | 1 | |
| | | baseline | DD | DD | DD | | | | | |
| | 1 | 0 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | 2 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| Using<br>mouth-<br>wash | 3 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | FQC | 2 | 0 | 0 | 1 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | 4 | baseline | DD | DD | DD | | | | | |
| | | 0 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |
| | | baseline | FQC | DD | DD | | | | 1 | |
| | 5 | 0 min | DD | FQC | DD | 2 | 0 | 0 | 1 | |
| | | 30 min | DD | DD | DD | 3 | 0 | 0 | 0 | |

{14}------------------------------------------------

{15}------------------------------------------------

FQC = Replicate failed QC according to QC criteria on the first run; FQC replicates were re-run on a separate day if saliva sample was available

Out of a total 225 replicates run, 32/225 failed OC in this study. Of these, 18 replicates did not meet the QC criterion "call rate ≥ 0.980". Another 3 replicates yielded DNA concentrations below the QC criterion of 15 ng/uL (all of these replicates were from a sample collected at the 0 minute time point). Another 11 replicates did not have sufficient saliva volume for automated DNA extraction.

All replicates that failed BeadChip QC on the original run and had sufficient saliva volume or DNA concentration ≥15 ng/uL (10/32 replicates) for a re-run passed QC acceptance criteria on the re-run (per laboratory SOPs) and yielded correct genotype calls. The results indicate that saliva samples should be collected at least 30 minutes after eating, drinking, chewing gum, or using mouthwash.

#### Smoking Interference Study

To evaluate the effects of smoking before saliva collection on the accuracy of the BeadChip assay for the BLMAsh genotype, a group of 5 donors provided 3 samples each (baseline/control sample taken at least 60 minutes prior to smoking, samples

{16}------------------------------------------------

collected immediately after smoking and samples collected 30 minutes after smoking). The smoking exogenous substance samples were tested in triplicate for a total of 45 test samples (5 donors x 1 condition x 3 time points x 3 replicates). To confirm the BeadChip genotype, each (6) TSCC sample was also sequenced by bidirectional Sanger sequencing. The smoking interference study also indicates that saliva samples should be collected at least 30 minutes after smoking. The passing criteria were a minimum of 95% concordant genotype calls across all individuals at each time point. Results are shown in the table below:

| Individual | Time Point | Replicates Genotype* | | |
|------------|------------|----------------------|-------------|-------------|
| | | replicate 1 | replicate 2 | replicate 3 |
| 1 | baseline | DD | DD | DD |
| | 0 min | DD | DD | DD |
| | 30 min | DD | DD | DD |
| 2 | baseline | DD | FQC | DD |
| | 0 min | DD | DD | DD |
| | 30 min | DD | DD | FQC |
| 3 | baseline | DD | DD | DD |
| | 0 min | DD | DD | DD |
| | 30 min | DD | DD | DD |
| 4 | baseline | DD | DD | DD |
| | 0 min | DD | FQC | DD |
| | 30 min | DD | DD | DD |
| 5 | baseline | DD | DD | DD |
| | 0 min | FQC | FQC | DD |
| | 30 min | DD | DD | DD |

Smoking Interference Study Genotype Call Raw Data

FOC = Sample failed OC according to OC criteria

*DD genotype=wild type homozygous non-carrier

Out of a total 45 replicates run, 5/45 failed QC in this study. Of these, 3 replicates did not meet the QC criterion "call rate ≥ 0.980". Another 2 replicates yielded DNA concentrations below the QC criterion of 15 ng/uL (both replicates were from a sample collected immediately after smoking). Out of the 5 samples that failed QC, 2 samples had DNA concentration <15 ng/uL, thus 3 samples that passed QC acceptance criteria on the re-run (per laboratory SOPs) yielded correct genotype calls. The results demonstrate that saliva samples should be collected at least 30 minutes after smoking.

## Microbial Interference Study

A microbial interference study was performed to determine whether microbial DNA affects successful assignment of the correct BLMAh genotype using the 23andMe
BeadChip assay. Six DNA samples taken from 14) Tsccacell lines were tested (4 BLMAsh homozygous common ("DD") samples/3 replicates, 1 BLMAsh heterozygous

{17}------------------------------------------------

("DI") sample/3 replicates and 1 BLMAsh homozygous rare ("II") sample/3 replicates). Genomic DNA from five microbes was spiked into an aliquot of DNA from each 64) Tscc sample, followed by BeadChip genotyping. An additional aliquot was spiked with buffer and served as control. Human and microbial DNA was diluted so that the DNA concentration in the final 50/50 mixture was within the analytical range. The minimum required DNA concentration was 15 ng/uL. To confirm the BeadChip genotype, each (6) Tsice sample was also sequenced by bidirectional Sanger sequencing. BeadChip genotypes were compared with sequenced genotypes to determine the rates of correct BeadChip genotype calls. The passing criteria were defined as a minimum of 95% correct genotype calls across all individual samples for each of the five microbe conditions. Results are shown in the table below:

| | | | Replicate Genotypes | | |
|--------------|-------------|----------------|---------------------|-------------|-------------|
| Genotype | Sample | Microbe | replicate 1 | replicate 2 | replicate 3 |
| BLMAsh<br>DD | b(4) TS/CCI | buffer control | DD | DD | DD |
| | | S. epidermidis | DD | DD | DD |
| | | S. mutans | DD | DD | DD |
| | | L. casei | DD | DD | DD |
| | | A. | DD | DD | DD |
| | | C. albicans | DD | DD | DD |
| BLMAsh<br>DD | b(4) TS/CCI | buffer control | DD | DD | DD |
| | | S. epidermidis | DD | DD | DD |
| | | S. mutans | DD | DD | DD |
| | | L. casei | DD | DD | DD |
| | | A. | DD | DD | DD |
| | | C. albicans | DD | DD | DD |
| BLMAsh<br>DD | b(4) TS/CCI | buffer control | DD | DD | DD |
| | | S. epidermidis | DD | DD | DD |
| | | S. mutans | DD | DD | DD |
| | | L. casei | DD | FOC | FOC |
| | | A. | DD | DD | DD |
| | | C. albicans | DD | DD | DD |
| BLMAsh<br>DD | b(4) TS/CCI | buffer control | DD | DD | DD |
| | | S. epidermidis | DD | DD | DD |
| | | S. mutans | DD | DD | FOC |
| | | L. casei | DD | DD | DD |
| | | A. | DD | DD | DD |
| | | C. albicans | DD | DD | DD |
| BLMAsh<br>DI | b(4) TS/CCI | buffer control | DI | DI | DI |
| | | S. epidermidis | DI | DI | DI |
| | | S. mutans | DI | DI | DI |
| | | L. casei | DI | DI | DI |
| | | A. | DI | DI | DI |

Microbial Interference Study Genotype Call Raw Data

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| | | C. albicans | DI | DI | DI |
|----------------|-------------|----------------|----|----|-----|
| BLMAsh<br>II * | | buffer control | NC | NC | NC |
| | | S. epidermidis | NC | NC | NC |
| | b(4) TS/CCI | S. mutans | NC | NC | FQC |
| | | L. casei | NC | NC | NC |
| | | A. | NC | NC | NC |
| | | C. albicans | NC | NC | NC |

*NC = No Call, as per protocol for BLMAsh II genotype samples.

FQC = Replicate failed QC according to QC criteria; FQC replicates were re-run on a separate day

Four replicates that failed OC in this study did not meet the OC criterion "call rate > 0.980". Upon re-running these samples, all replicates produced correct genotype calls.

- f. Assay Cut-off:
Not applicable.

- Specimen Stability at 2-8° C g.
Saliva samples for testing are collected with the Oragene Dx OGD-500.001 collection device. See k141410 for sample stability information.

- h. Shipping Stability
Saliva samples are shipped for testing in the Oragene Dx OGD-500.001 collection device. See k141410 for sample shipping stability information.

- 2. Comparison Studies:
- Method Comparison with Predicate Device: a.

Accuracy was evaluated by the agreement of the genetic variant determinations by this test with bi-directional sequencing results.

Saliva samples were randomly selected from the 23andMe Biobank, in which saliva samples were collected using the DNA Genotek OrageneDx 500.001 collection device. Saliva sample selection was blinded to previously determined genotypes and at least 20 carrier samples (DI, as detected by genotyping) were selected. A total of 65 saliva samples were selected for the study (25 DD and 22 DI samples tested at Site 1; 18 DI samples samples tested at Site 2) in addition to 6 human cell line samples tested (4 DD samples, 1 DI sample and 1 II sample tested at both sites). All 71 samples were sequenced using bi-directional sequencing. The comparison study was conducted at 2 sites; results of the test were compared with sequencing results. If a replicate fails QC ("FQC") criteria on the first run, the replicate was re-run once using the same sample. Five saliva samples failed QC at Site 1 and 1 saliva sample failed QC at the Site 2. All re-run samples produced correct genotype results. Study

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| Summary of Comparison Data of Saliva Samples for <span style="font-size: smaller;">b(4) TS/CCI</span> | | | | |
|-------------------------------------------------------------------------------------------------------|------------------------------------|---|---------------------------|------|
| | | | Bi-directional sequencing | |
| | | | "DI" | "DD" |
| PGS<br>Carrier<br>test<br>for<br>Bloom<br>Syndrome | First run<br>"1 Variant Detected" | | 21 | 0 |
| | First run<br>"0 Variants Detected" | | 0 | 21 |
| First<br>Run<br>FQC<br>Result | Re-run<br>"1 Variant<br>Detected" | 1 | 0 | |
| | | | | |
| | | | | |
| | Re-run<br>"0 Variants<br>Detected" | 0 | 4 | |
| | | | | |
| | Re-run<br>FQC | | 0 | 0 |
| Total | | | 22 | 25 |

results and % agreement are provided in the tables below:

Summary of Comparison Data of Saliva Samples for b(4) TS/CCI

| | | Bi-directional sequencing | | |
|----------------------------------------------------|-------------------------------|------------------------------------|------|---|
| | | "DI" | "DD" | |
| PGS<br>Carrier<br>test<br>for<br>Bloom<br>Syndrome | First run | "1 Variant Detected" | 17 | 0 |
| | | "0 Variants Detected" | 0 | 0 |
| | First<br>Run<br>FQC<br>Result | Re-run<br>"1 Variant<br>Detected" | 1 | 0 |
| | | Re-run<br>"0 Variants<br>Detected" | 0 | 0 |
| | | Re-run<br>FQC | 0 | 0 |
| | | Total | | |

The homozygous common (DD) genotype results at both sites did not produce results for the "1 Variant Detected Category" and vice versa for the heterozygous variant genotype regarding the "0 Variants Detected" category. All samples that failed QC on the first run produced correct genotype results upon re-running them at both sites.

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| | Positive Percent Agreement<br>Saliva Samples with “DI” by<br>bi-directional sequencing | | Negative Percent Agreement<br>Saliva Samples with “DD” by<br>bi-directional sequencing | |
|----------|----------------------------------------------------------------------------------------|--------------------------|----------------------------------------------------------------------------------------|--------------------------|
| | Percent correct<br>results | %FQC<br>on the first run | Percent correct<br>results | %FQC<br>on the first run |
| Site 1 | 100% (22/22) | 4.5% (1/22) | 100% (25/25) | 16.0% (4/25) |
| Site 2 | 100% (18/18) | 5.6% (1/18) | n/a | n/a |
| Combined | 100% (40/40) | 5.0% (2/40) | 100% (25/25) | 16.0% (4/25) |

#### Positive and Negative Percent Agreements for Saliva Samples for Both Sites

Results for the cell line samples tested at both sites are as follows:

- i) The DI sample had correct genotyping results on the first run at both sites;
- ii) All 4 DD samples had correct genotyping on the first run at both sites;

iii) The II sample failed QC on the first run at Site 2 and produced a correct (no call) result upon re-running the sample.

The following table presents PPA and NPA for saliva and human cell line samples combined.

| | Percent of<br>correct calls | 95% CI |
|-------------------------------------|-----------------------------|-----------------|
| Positive Percent<br>Agreement (PPA) | 100% (41/41) | 91.4% to 100%* |
| Negative Percent<br>Agreement (NPA) | 100% (29/29) | 88.3% to 100%* |
| Overall Agreement | 100% (70/70) | 96.3% to 100%** |

#### PPA and NPA for Saliva and Human Cell Line Samples Combined for Both Sites

*95% two-sided confidence interval

** 95% one-sided confidence interval

Overall agreement was 100% (70/70) with 95% confidence interval of 96.3% to 100%.

- b. Matrix Comparison:
Not applicable. This test is for use with human saliva samples only.

- 3. Clinical Studies:
## Clinical Performance

The BLMAsh variant covered by this test is mainly found in people of Ashkenazi Jewish descent. Approximately 1 in 107 people (0.93%) with this ethnicity carries this variant. The BLMAsh variant is rare and not well studied in other ethnic groups.

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| Ancestry Group | Frequency | Number of Tested |
|------------------|-----------|------------------|
| Ashkenazi Jewish | 1.03% | b(4) TS/CC |
| European | 0.02% | |
| Latino | <0.04% | |
| African American | 0.00 | |
| Asian | 0.00 | |

# Carrier Frequency of the BLMAsh Mutation in the 23andMe Database

More than 99% of all patients of individuals of Ashkenazi Jewish descent known to have Bloom syndrome have two copies of this mutation12; therefore, this test is expected to detect more than 99% of Bloom syndrome carriers in people of Ashkenazi Jewish descent.

# Pre-test and Post-test Carrier Risks for Different Results of PGS test for Bloom Syndrome

| | Ashkenazi Jewish | Other Ancestry Groups |
|------------------------------------------------------------------------------------|------------------|-----------------------|
| BLMAsh frequency among<br>patients with Bloom syndrome | >99%1 | Unknown |
| Pre-test carrier risk | 1 in 1071 | Likely < 1 in 107 |
| Carrier risk for result<br>“1 Variant Detected”<br>of PGS test for Bloom Syndrome | 99% | 50%-99%* |
| Carrier risk for result<br>“0 Variants Detected”<br>of PGS test for Bloom Syndrome | < 1 in 11,000 | Likely < 1 in 107 |

* The carrier risk depends on subject ethnicity; for some ethnicities, this risk can be lower than 50%.

## References:

- 1. Gross, S.J., Pletcher, B.A., Monaghan, K.G. (2008). ACMG Practice Guidelines: Carrier screening in individuals of Ashkenazi Jewish descent. Genet Med. 10(1):54-56.
- 2. German, J., Sanz, M.M. Syndrome-Causing Mutations of the BLM Gene in Persons in the Bloom's Syndrome Registry. Hum Mut. (2007) 28(8):743-753.

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#### Other clinical supportive data (when a. and b. are not applicable):

A user comprehension study was performed in order to assess comprehension of the proposed labeling of the PGS Test report in a demographically diverse sample and to evaluate potential factors influencing comprehension. Specifically, the user studies were performed in order to assess user comprehension of representative test reports. Furthermore, the studies were comprised of naïve participants representative of a broad intended use population, and conducted in a controlled, lab-based setting so that participant experiences with the survey could be recorded in detail.

The user comprehension study used quota-based sampling to recruit a naïve sample of participants that were demographically diverse according to age, race/ethnicity, and education level. The study was conducted at 5 locations across the U.S. A target of at least 100 subjects were tested across each of 5 different representative test reports for the PGS (Full Ashkenazi Jewish combined with either variant present, variant absent, not determined; partial Ashkenazi Jewish combined with variant absent and no Ashkenazi Jewish and variant absent) with each location recruiting at least 120 participants to achieve this target. A total of 11 of 678 (1.6%) participants were excluded after enrollment for the following reasons: careless responders (indicating an incorrect answer on a main survey question with an obvious correct answer), error in data recording, previous 23andMe study participants.

Participants were assigned to study arms at each facility in the order in which they started their session. Participants were asked to complete the following online tasks:

a. Background survey - familiarity with genetics and interest in genetic testing b. Pre-test comprehension survey - comprehension of concepts assessed in the main comprehension survey

c. Comprehension survey instructions and education module (view only) description of the main survey task and explanation of genetic testing concepts d. Main comprehension survey - Representative Bloom Syndrome Test report with results for a fictional individual, paired with comprehension survey e. Post-test usability survey - participant's general feedback about the Bloom Syndrome Test report and the survey experience

Primary comprehension assessment addressed the following comprehension concepts: purpose of the PGS, limitations of the test (variants covered), relevant ethnicities for the test, meaning of test results, and appropriate follow-up actions. Secondary analyses included assessment of participants' baseline knowledge of genetic testing concepts, survey completion rates, and evaluation of qualitative feedback from participants and moderators. The table below demonstrates the results for each study arm and testing concept:

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| Comprehension<br>concept | Comprehension rates (%) within each study arm | | | | | Overall<br>comprehension<br>rates (%) |
|--------------------------|-----------------------------------------------|-------------------------------|------------------------------|------------------------------------|----------------------------|---------------------------------------|
| | Full Match<br>Variant Absent | Full Match<br>Variant Present | Full Match<br>Not Determined | Partial<br>Match<br>Variant Absent | No Match<br>Variant Absent | |
| Purpose of test | 90.4 | 95.5 | 94.8 | 91.7 | 89.2 | 92.4 |
| Test results | | 95.5 | 94.8 | | | 95.2 |
| Limitations of test | 88.9 | 91.8 | 94.1 | 93.2 | 90.0 | 91.6 |
| Ethnicity relevance | 96.3 | | 95.6 | 91.7 | 94.6 | 94.6 |
| Meaning of results | 89.6 | 97.8 | | 94.0 | 87.7 | 92.3 |
| Appropriate<br>follow-up | 94.1 | 95.5 | 94.1 | 92.5 | 92.3 | 93.7 |

The overall comprehension rates across all study arms for each comprehension concept ranged from 91.6% to 95.2%. Overall comprehension rates were above 90% for all comprehension concepts across all study arms.

#### 4. Expected values/Reference range:

Testing for the BLMAsh mutation is expected to have clinical sensitivity (test coverage) greater than 99% in Ashkenazi Jewish individuals (Gross et al., 2008). Approximately 0.75-1% of individuals with Aslikenazi Jewish ancestry carry the mutation (Peleg et al., 2002, Gross et al., 2008; Scott et al., 2010), which is consistent with the observed frequency in 23andMe customers of Ashkenazi Jewish descent (see table below). The frequency of this mutation in other populations has not been reported and it is assumed to be much rarer in non-Jewish populations.

| Ancestry Group | Frequency | Number of Tested<br>Individuals |
|------------------|-----------|---------------------------------|
| Ashkenazi Jewish | 1.03% | b(4) TS/CC |
| European | 0.02% | |
| Latino | <0.04% | |
| African American | 0.00 | |
| Asian | 0.00 | |

#### Carrier Frequency of the BLMash Mutation in the 23andMe Database

More than 99% of all patients of individuals of Ashkenazi Jewish descent known to have Bloom syndrome have two copies of this mutation (Gross et al., 2008, German et al., 2007); therefore, this test is expected to detect more than 99% of Bloom syndrome…