AlphaID™ At Home Genetic Health Risk Service

K221420 · Progenika Biopharma S.A., A Grifols Company · PTA · Oct 27, 2022 · Immunology

Device Facts

• Record ID: K221420
• Device Name: AlphaID™ At Home Genetic Health Risk Service
• Applicant: Progenika Biopharma S.A., A Grifols Company
• Product Code: PTA · Immunology
• Decision Date: Oct 27, 2022
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5950
• Device Class: Class 2
• Attributes: Software as a Medical Device, Real-World Evidence

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K221420 · Oct 27, 2022	AlphaID™ At Home Genetic Health Risk Service	Progenika Biopharma S.A., A Grifols Company	Published clinical literature; Meta-analyses of clinical cases	The sponsor used published clinical case data to define and validate the risk categories (Increased, Slightly increased, Not likely at increased risk, Unknown) for 120 possible genetic variant combinations associated with AATD.	AATD; SERPINA1; Risk categorization; Clinical case literature

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Literature-based risk categorization; Retrospective analysis of published clinical cases; Follow-up/Duration: Not applicable; Study Period: Not applicable	Individuals with SERPINA1 gene variants and clinical outcomes (lung/liver disease); Sample Size: Variable (based on meta-analyses of multiple studies); Number of Sites: Not applicable	Not applicable for this study	Risk of developing lung and/or liver disease based on genetic variant combination

Indications for Use

The AlphaID™ At Home Genetic Health Risk Service uses qualitative genotyping to detect clinically relevant genetic variants associated with alpha-1 antitrypsin deficiency (AATD) in genomic DNA isolated from human saliva collected from individuals ≥ 18 years with ORAcollect·Dx OCD-100.014 for the purpose of reporting and interpreting Genetic Health Risks (GHR). This Service is indicated for reporting 14 genetic variants in the SERPINA1 gene: PI*S; PI*Z; PI*I; PI*M procida; PI*M malton; PI*S iiyama; PI*Q0 granite falls; PI*Q0 west; PI*Q0 bellingham; PI*F; PI*P lowell; PI*Q0 mattawa; PI*Q0 clayton, and PI*M heerlen. The report describes if a person is at an increased risk of developing either lung and/or liver disease linked to AATD. The report does not describe a person's overall risk of developing lung and/or liver disease. AATD is more common in persons of European descent.

Device Story

Service uses saliva samples collected via ORAcollect-Dx OCD-100.014; shipped to CLIA-certified lab. Genomic DNA extracted; amplified/biotinylated via multiplex PCR; hybridized to allele-specific oligonucleotide probes on color-coded microspheres. Luminex 200 system detects fluorescent signal. A1AT Genotyping Test Analysis Software processes raw fluorescence data to determine genotypes; maps to specific Genetic Health Risk (GHR) report templates. Consumers access personalized reports via web portal after completing educational module. Reports categorize risk for lung/liver disease (Increased, Slightly increased, Not likely at increased, Unknown) based on variant combinations. Benefits include awareness of genetic predisposition to AATD-linked conditions; facilitates informed discussions with healthcare professionals.

Clinical Evidence

Bench testing only. Method comparison study (n=227) against bi-directional sequencing showed 100% agreement (95% CI: 98.3-100%). Reproducibility study (n=110) showed 100% concordance between labs. User comprehension study (n=525) demonstrated 94.0-99.5% comprehension across domains. Interference testing (endogenous, exogenous, microbial) showed no impact on performance.

Technological Characteristics

Uses Luminex xMAP technology; multiplex PCR amplification; allele-specific oligonucleotide probes on color-coded microspheres. Requires 384-well/96-well Veriti Dx thermal cyclers and Luminex 200 instrument. Software: A1AT Genotyping Test Analysis Software (genotype calling) and web portal (report generation).

Indications for Use

Indicated for individuals ≥18 years old to detect 14 SERPINA1 gene variants associated with AATD risk for lung/liver disease. Not for diagnosis, medical treatment, or health status assessment. Requires confirmatory testing for clinical decisions.

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.

Predicate Devices

• 23andMe Personal Genome Service (PGS) Genetic Health Risk Test (Alpha-1 Antitrypsin Deficiency) (DEN160026)

Submission Summary (Full Text)

{0}

**FDA** U.S. FOOD & DRUG
ADMINISTRATION

# **510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY ONLY**

# **I Background Information:**

# **A 510(k) Number**

K221420

# **B Applicant**

Progenika Biopharma S.A.

# **C Proprietary and Established Names**

AlphaID At Home Genetic Health Risk Service

# **D Regulatory Information**

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PTA | Class II | 21 CFR 866.5950 - Genetic Health Risk Assessment System | IM - Immunology |

# **II Submission/Device Overview:**

# **A Purpose for Submission:**

New device

# **B Measurand:**

14 SERPINA1 gene variants in genomic DNA obtained from a human saliva sample

# **C Type of Test:**

The AlphaID At Home Genetic Health Risk Service determines if a person has variants associated with a higher risk of developing alpha-1 antitrypsin deficiency (AATD) associated lung or liver disease. The Service is intended to provide the AATD risk report. The report is based on a qualitative genetic test for detecting 14 genetic variants in the serpin peptidase

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

{1}

inhibitor class A member 1 (SERPINA1) gene: PI*S; PI*Z; PI*I; PI*M procida; PI*M malton; PI*S iiyama; PI*Q0 granite falls; PI*Q0 west; PI*Q0 bellingham; PI*F; PI*P lowell; PI*Q0 mattawa; PI*Q0 clayton, and PI*M heerlen. The AlphaID At Home Genetic Health Risk Service is for over-the-counter use.

### III Intended Use/Indications for Use:

#### A Intended Use(s):

See Indications for Use below.

#### B Indication(s) for Use:

The AlphaID At Home Genetic Health Risk Service uses qualitative genotyping to detect clinically relevant genetic variants associated with alpha-1 antitrypsin deficiency (AATD) in genomic DNA isolated from human saliva collected from individuals ≥ 18 years with ORAcollect-Dx OCD-100.014 for the purpose of reporting and interpreting Genetic Health Risks (GHR).

This Service is indicated for reporting 14 genetic variants in the SERPINA1 gene: PI*S; PI*Z; PI*I; PI*M procida; PI*M malton; PI*S iiyama; PI*Q0 granite falls; PI*Q0 west; PI*Q0 bellingham; PI*F; PI*P lowell; PI*Q0 mattawa; PI*Q0 clayton, and PI*M heerlen. The report describes if a person is at an increased or decreased risk of developing either lung and/or liver disease linked to AATD. The report does not describe a person's overall risk of developing lung and/or liver disease. AATD is more common in persons of European descent.

#### C Special Conditions for Use Statement(s):

- • For over-the-counter (OTC) use.
- • The test is intended for users ≥18 years old.
- • The test is not a substitute for an appointment with a healthcare professional. It is recommended that the user consults with a healthcare professional if the user has any questions or concerns about his/her results.
- • The test does not diagnose a disease or condition, determine medical treatment or other medical intervention, or tell the user anything about their current state of health. Only a healthcare professional can diagnose a disease or condition.
- • Any diagnostic or treatment decisions must be based on confirmatory prescription testing and/or other information that a healthcare professional determines to be appropriate for the patient, such as additional clinical testing and other risk factors that may affect individual risk and health care.
- • The test detects 14 variants in the SERPINA1 gene linked to AATD. These 14 variants explain 95% of AATD cases. The absence of a variant tested does not rule out the presence of other genetic variants that may be disease-related.
- • The test does not describe a person's overall risk of developing AATD. In addition, other genetic and all non-genetic factors should be considered
- • The laboratory may be unable to process every user's sample. The probability that the laboratory cannot process a sample can be up to 0.5%. If this happens, the user will receive an

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email notification. The user will also receive another AlphaID At Home Saliva Collection Kit to provide a new sample to the laboratory.

- The user's race, ethnicity, age, and sex may affect how the genetic results are interpreted.
- Subject to meeting limitations contained in the special controls under the regulation 21 CFR 866.5950.

# D Special Instrument Requirements:

The AlphaID At Home Genetic Health Risk Service is to be performed using 384-well Veriti Dx and 96-well Veriti Dx thermal cyclers, and the Luminex 200 instrument.

# IV Device/System Characteristics:

# A Device Description:

The AlphaID At Home Genetic Health Risk Service uses qualitative genotyping to detect clinically relevant genetic variants associated with alpha1-antitrypsin deficiency (AATD) in genomic DNA isolated from human saliva and provides a report describing if a person is at risk of developing either lung and/or liver disease linked to AATD. This Service is direct-to-consumer and intended for an over-the-counter (OTC) use.

The AlphaID At Home Genetic Health Risk Service is composed of:

(i) ORAcollect-Dx OCD-100.014 (cleared under K212745) is intended for use in the non-invasive collection of saliva samples.
(ii) A1AT Genotyping Test (cleared under K192858) is intended for the genetic analysis and detection of genetic variants associated with alpha-1 antitrypsin deficiency (AATD) using the A1AT Genotyping Test ANALYSIS SOFTWARE, and
(iii) AlphaID At Home Genetic Health Risk Service website and result portal software are intended to provide the contents and the procedure to order and use the OTC Service.

A consumer's saliva is self-collected using ORAcollect-Dx OCD-100.014 manufactured by DNA Genotek, Inc which consists of a collection tube containing a stabilizing buffer solution. Once the sample is collected, it is shipped to a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory for processing.

Human DNA from the saliva sample is isolated, processed and analyzed with the A1AT Genotyping Test that is indicated for reporting 14 genetic variants in the SERPINA1 gene: PI*S; PI*Z; PI*I; PI*M procida; PI*M malton; PI*S iiyama; PI*Q0 granite falls; PI*Q0 west; PI*Q0 bellingham; PI*F; PI*P lowell; PI*Q0 mattawa; PI*Q0 clayton, and PI*M heerlen. Briefly, genomic DNA extracted from human saliva is amplified and biotinylated by multiplex polymerase chain reaction (PCR) and the resulting labeled PCR products are denatured and hybridized to allele-specific oligonucleotide probes coupled to color-coded microspheres. Hybridized DNA is labeled with a fluorescent conjugate and the resulting signal is detected with a Luminex 200 system. Raw fluorescence data is processed with the A1AT Genotyping Test ANALYSIS SOFTWARE to provide allelic variant genotypes, which are subsequently converted into associated alleles. Additionally, the software application also provides the type of

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Genetic Health Risk Report associated with the identified alleles, which is subsequently used as the basis for the generation of personalized reports by the AlphaID At Home Genetic Health Risk Service website and result portal.

Depending on the specific variant combination detected, the AlphaID At Home Genetic Health Risk Service provides the individuals' genetic health risk for developing lung and liver disease linked to AATD. Personalized reports, in an easy-to-understand format, are generated for each consumer that help to understand the meaning of their results and any appropriate actions that may be taken based on their results. In addition, every user before receiving report must undergo the educational module that helps to understand the AlphaID At Home Genetic Health Risk Service results report and explain how genetics can impact a person's health risk for lung disease and/or liver disease linked to AATD. The AlphaID At Home Genetic Health Risk Service uses four (4) categories summarized in the table below to define risk. One of these risk categories has been assigned to each genetic result based on the information gathered from reported clinical cases for each genetic result. If any of the 14 variants tested by this Service is detected, one of the following four risk categories will be reported:

| Risk Categories | |
| --- | --- |
| Increased risk | Above 80% of people with this genetic result develop lung or liver disease during their lifetime. |
| Slightly increased risk | 20-80% of people with this genetic result develop lung or liver disease during their lifetime. |
| Not likely at increased risk | Below 20% of people with this genetic result develop lung or liver disease during their lifetime. |
| Unknown risk | More clinical studies are needed to determine the risk level associated with this genetic result |

When no variants are detected, the report shows '*Not Likely at Risk*' for AATD. When the service is not able to provide a genetic result, the associated lung and liver disease risk categories cannot be determined, and another type of report will be generated: '*Variant not determined*'.

Based on the number of variants detected (0, 1, 2, or variant not determined) and specific lung and liver disease risk categorization reported, a total of 15 types of reports can be generated by the AlphaID At Home Genetic Health Risk Service, which are shown in the table below:

| Type of Report | Number of variants | Reported Lung Disease Risk Category | Reported Liver Disease Risk Category | Number of genetic results | Frequency of genetic results, % |
| --- | --- | --- | --- | --- | --- |
| 1 | 0 | Not likely at risk for AATD | Not likely at risk for AATD | 1 | 94.95 |
| 2 | 1 | Not likely at increased risk | Not likely at increased risk | 11 | 4.23 |
| 3 | 1 | Not likely at increased risk | Slightly increased risk | 1 | 0.002 |
| 4 | 1 | Not likely at increased risk | Unknown risk | 1 | 0.002 |
| 5 | 1 | Not likely at increased risk (never-smokers) Slightly increased risk (ever-smokers) | Slightly increased risk | 1 | 0.780 |
| 6 | 2 | Not likely at increased risk | Not likely at increased risk | 1 | 0.40 |
| 7 | 2 | Slightly increased risk | Not likely at increased risk | 1 | <0.00001 |
| 8 | 2 | Slightly increased risk | Slightly increased risk | 5 | 0.017 |

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| Type of Report | Number of variants | Reported Lung Disease Risk Category | Reported Liver Disease Risk Category | Number of genetic results | Frequency of genetic results, % |
| --- | --- | --- | --- | --- | --- |
| 9 | 2 | Slightly increased risk | Unknown risk | 1 | <0.00001 |
| 10 | 2 | Increased risk | Not likely at increased risk | 15 | <0.00001 |
| 11 | 2 | Increased risk | Slightly increased risk | 7 | 0.0016 |
| 12 | 2 | Unknown risk | Slightly increased risk | 15 | 0.0001 |
| 13 | 2 | Unknown risk | Not likely at increased risk | 29 | 0.0001 |
| 14 | 2 | Unknown risk | Unknown risk | 31 | 0.0069 |
| 15 | Variant not determined | Not determined risk | Not determined risk | N/A | N/A |

### B Principle of Operation:

The AlphaID At Home Genetic Health Risk Service is performed by a CLIA-certified laboratory using the Alpha-1 antitrypsin (A1AT) Genotyping Test.

The A1AT Genotyping Test utilizes Luminex xMAP technology. Genomic DNA is extracted from human saliva samples collected as buccal swabs using ORAcollect-Dx OCD-100.014. Extracted DNA is amplified and biotinylated by multiplex PCR and the PCR products are denatured and hybridized to oligonucleotide probes coupled to color-coded beads. Hybridized DNA is labeled with fluorescent conjugate and the resulting signal is detected with a Luminex 200 system (with xPONENT software). Raw data obtained is processed with the A1AT Genotyping Test Analysis Software.

The A1AT Genotyping Test Analysis Software algorithm converts the allelic variant genotypes into associated alleles. Additionally, this software application also provides an associated template number for each sample, this is, a code that defines the type of Genetic Health Risk Report that is to be generated for each individual, depending on its combination of variants. This template number will be subsequently used as the basis for the generation of personalized reports by the AlphaID System.

### V Substantial Equivalence Information:

### A Predicate Device Name(s):

23andMe Personal Genome Service (PGS) Genetic Health Risk Report for Alpha-1 Antitrypsin Deficiency

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

DEN160026

### C Comparison with Predicate(s):

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| Device & Predicate Device(s): | Device K221420 | Predicate DEN160026 |
| --- | --- | --- |
| Device Trade Name | AlphaID At Home Genetic Health Risk Service | 23andMe Personal Genome Service (PGS) for A1AT |
| **General Device Characteristic Similarities** | | |
| Intended Use/ Indications For Use | The AlphaID At Home Genetic Health Risk Service uses qualitative genotyping to detect clinically relevant genetic variants associated with alpha-1 antitrypsin deficiency (AATD) in genomic DNA isolated from human saliva collected from individuals ≥ 18 years with ORACollect-Dx OCD-100.014 for the purpose of reporting and interpreting Genetic Health Risks (GHR). The AlphaID At Home Genetic Health Risk Service is indicated for reporting 14 genetic variants in the *SERPINA1* gene: PI*S; PI*Z; PI*I; PI*M procida; PI*M malton; PI*S iiyama; PI*Q0 granite falls; PI*Q0 west; PI*Q0 bellingham; PI*F; PI*P lowell; PI*Q0 mattawa; PI*Q0 clayton, and PI*M heerlen. The report describes if a person is at an increased or decreased risk of developing either lung and/or liver disease linked to AATD. The report does not describe a person's overall risk of developing lung and/or liver disease. AATD is more common in persons of European descent. | 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 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. |
| Special Conditions for Use Statements | a. For over-the-counter (OTC) use. b. The test is intended for users ≥18 years old. c. The test is not a substitute for an appointment with a healthcare professional. It is recommended that the user consult with a healthcare professional if the user have any questions or concerns about his/her results. d. The test does not diagnose a disease or condition, determine medical treatment or other medical intervention, or tell the user anything | a. For over-the-counter (OTC) use. b. 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. 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 |

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| | about their current state of health. Only a healthcare professional can diagnose a disease or condition. e. Any diagnostic or treatment decisions must be based on confirmatory prescription testing and/or other information that a healthcare professional determines to be appropriate for the patient, such as additional clinical testing and other risk factors that may affect individual risk and health care. f. The test detects 14 variants in the SERPINA1 gene linked to AATD. These 14 variants explain 95% of AATD cases. The absence of a variant tested does not rule out the presence of other genetic variants that may be disease-related. g. The test does not describe a person's overall risk of developing AATD. In addition, other genetic and all non-genetic factors should be considered h. The laboratory may be unable to process every user's sample. The probability that the laboratory cannot process a sample can be up to 0.5%. If this happens, the user will receive an email notification. The user will also receive another AlphaID At Home Saliva Collection Kit to provide a new sample to the laboratory. i. The user's race, ethnicity, age, and sex may affect how the genetic results are interpreted. j. Subject to meeting limitations contained in the special controls under the regulation 21 CFR 866.5950 | 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. e. The test does not diagnose any specific health conditions. Results should not be used 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. |
| --- | --- | --- |
| Type of Test | Qualitative genotyping for allelic variant determination through single nucleotide polymorphism detection | Same |
| Design | Software application that includes product information page, e-commerce (registration and order DNA kit), secure login, download genetic report | Same |

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| Interpretation of results | For over-the-counter use (OTC). Specialized interpretation by a physician not required | Same |
| --- | --- | --- |
| Specimen Type | Saliva | Same |
| Measurand | Genomic DNA | Same |
| Comparison with Sanger Bidirectional Sequencing | Overall agreement for the 14 variants was 100%. | Overall agreement for the two variants was 100%. |
| Human factors | User Comprehension Study | Same |
| **General Device Characteristic Differences** | | |
| Specimen Collection Kit | DNA Genotek Inc., ORAcollect Dx OCD-100.014 | DNA Genotek Inc., Oragene Dx model OGD-500.001 |
| Technology | Polymerase Chain Reaction (PCR) amplification and hybridization based Luminex technology | Multiplex customized genotyping chip (BeadChip v4 assay) |
| Method and Sequencing Platform | Multiplex polymerase chain reaction (PCR) and multiplex Allele Specific Primer Extension (ASPE) with Luminex's Universal Tag sorting system on the Luminex 200 xMAP platform with xPONENT software and A1AT Genotyping Test Analysis software for the simultaneous detection and identification of allelic variants and their associated alleles. | Magnetic bead DNA extraction is performed using Tecan Evo. Genotyping and PCR analysis is conducted using a chip-based method with Illumina Infinium's BeadChip v4 assay, the Illumina iScan System for detection and analysis is facilitated with the use of GenomeStudio and Coregen software. |
| Special Instrument Requirements | Luminex 200 instrument (with xPONENT® software) The A1AT Genotyping Test ANALYSIS SOFTWARE processes raw data from the Luminex System (csv. files containing the MFI value for each bead type) to provide allelic variant genotypes which are subsequently converted into associated alleles, and the type of Genetic Health Risk Report associated with the identified alleles | 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. |
| Allelic Variants Detected | 14 allelic variants in the SERPINA1 gene: PI*I; PI*M procida; PI*M malton; PI*S iiyama; PI*Q0 granite falls; PI*Q0 west; PI*Q0 bellingham; PI*F; PI*P lowell; PI*Q0 mattawa; | Two allelic variants in the SERPINA1 gene: P*Z and P*S |

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| | PI*Q0 clayton, PI*M heerlen). | |
| --- | --- | --- |
| Analytical Sensitivity | The performance requirement for the A1AT Genotyping Test has been set at a minimum of 0.0215 ng/μL DNA. | The performance requirement for the PGS has been set at a minimum of 15 ng/μL DNA and maximum of 50 ng/μL DNA. |

### VI Standards/Guidance Documents Referenced:

- CLSI guideline EP07-A2, “Interference Testing in Clinical Chemistry; Approved Guideline–Second Edition”
- Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices (2005)
- General Principles of Software Validation Guidance for Industry and FDA Staff (2002)
- Off-The-Shelf Software Use in Medical Devices Guidance for Industry and Food and Drug Administration Staff (2019)
- Content of Premarket Submissions for Management of Cybersecurity in Medical Devices (2014)
- ISO 14971 Third Edition 2019-12 Medical devices - Application of risk management to medical devices

### VII Performance Characteristics (if/when applicable):

#### A Analytical Performance:

All results presented below met the sponsor’s pre-defined acceptance criteria outlined in the Special Controls of 21 CFR 866.5950. Information regarding samples that failed quality control (FQC) was also evaluated and presented in each study below.

##### 1. Precision/Reproducibility:

Refer to K171868 for results of the reproducibility study that was conducted with a total of 17 samples. The reproducibility of the assay was evaluated at three sites, with two operators at each site performing one run per day across three non-consecutive days (three runs per operator or six runs per site). Within a given run, the sample panel was tested in duplicate. Concordance with the expected results was 100% at the three sites.

Refer to K192858 for DNA extraction variability in saliva samples that was conducted using three different extraction methods: QIAamp DNA Blood Mini Kit (Qiagen), Commercial lysis and neutralization solutions (Sigma) and QIAsymphony DNA Mini Kit (Qiagen).

Refer to K171868 and K211115 for results of the lot-to-lot imprecision study.

##### 2. Linearity:

Not applicable

##### 3. Analytical Specificity/Interference:

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i. Interference

Refer to K192858 for results of studies conducted to evaluate the potential interference of substances usually found in saliva samples.

ii. Cross-reactivity

Refer to K171868. An In Silico DNA sequence analysis of A1AT Genotyping Test primers and probes confirmed sequence specificity and the absence of cross-reactivity.

iii. Cross-contamination

Refer to K171868

4. Assay Reportable Range:

Not applicable

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

i. Reagent stability:

Refer to K192858. The claimed reagent stability is 24 months when stored at 2–8°C and up to 9 months after the kit vials were first opened.

ii. Stability of collection device and specimens:

Saliva samples for testing are collected with the ORAcollect·Dx OCD-100.014 (customized version of ORAcollect·Dx OCD-100). Refer to K152464 for pre-collection shelf-life stability of the collection device, stability of samples post-saliva collection, and freeze-thaw stability of samples stored in the Oragene Dx collection device.

6. Detection Limit:

Refer to K211115 for results of the Lower Limit of Detection (LLoD) study that evaluated the impact of different levels of DNA input on test performance.

7. Assay Cut-Off:

Not applicable

# **B Comparison Studies:**

1. Comparison with Sanger Bidirectional Sequencing:

A method comparison study was performed with saliva samples collected in ORAcollect·Dx OCD-100.014 (customized version of ORAcollect·Dx OCD-100) to assess the accuracy of the AlphaID At Home Genetic Health Risk Service to correctly detect the genetic variants. A

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total of 227 samples representing all genetic variants interrogated by the assay were analyzed and compared with bi-directional Sanger sequencing (reference method).

The percent agreements (PAs) per allelic variant from clinical samples and contrived samples (8 synthetic DNAs samples) were calculated separately. The PA per allelic variant between the two methods was 100% for both clinical samples and contrived samples. The percentage of “No call” or “Invalid test” per allelic variant was also calculated and was 0% for both clinical samples and contrived samples.

The PAs of AlphaID At Home Genetic Health Risk Service genotype results with comparator results are summarized in the tables below.

| PI*I (c.187C>T) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 203 | 0 | 0 |
| C/T (heterozygous) | 0 | 15 | 0 |
| T/T (homozygous rare) | 0 | 0 | 1 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=219 | 203 | 15 | 1 |
| PA (C/C | C/C) = 100% (203/203) with 95% CI: 98.1%–100% PA (C/T | C/T) = 100% (15/15) with 95% CI: 79.6%–100% PA (T/T | T/T) = 100% (1/1) with 95% CI: 20.7%–100% Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%–1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 4 | 0 | 0 |
| C/T (heterozygous) | 0 | 1 | 0 |
| T/T (homozygous rare) | 0 | 0 | 3 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=8 | 4 | 1 | 3 |
| PA (C/C | C/C) = 100% (4/4) with 95% CI: 51.0%–100% PA (C/T | C/T) = 100% (1/1) with 95% CI: 20.7%–100% PA (T/T | T/T) = 100% (3/3) with 95% CI: 43.9%–100% Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%–2.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 207 | 0 | 0 |
| C/T (heterozygous) | 0 | 16 | 0 |
| T/T (homozygous rare) | 0 | 0 | 4 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=227 | 207 | 16 | 4 |
| PA (C/C | C/C) = 100% (207/207) with 95% CI: 98.2%–100% PA (C/T | C/T) = 100% (16/16) with 95% CI: 80.6%–100% PA (T/T | T/T) = 100% (4/4) with 95% CI: 51.0%–100% Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%–1.7%. |

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Minor allele frequency for PI*I SERPINA1 in the 1000 Genomes Project was 0.06%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of C/T and T/T are 99.128% and 99.997% correspondingly

| PI*M procida variant (c.194T>C) of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| T/T | T/C | C/C |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 211 | 0 | 0 |
| C/T (heterozygous) | 0 | 5 | 0 |
| T/T (homozygous rare) | 0 | 0 | 3 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=219 | 211 | 5 | 3 |
| PA (C/C | C/C) = 100% (211/211) with 95% CI: 98.2%-100% |
| PA (C/T | C/T) = 100% (5/5) with 95% CI: 56.6%-100% |
| PA (T/T | T/T) = 100% (3/3) with 95% CI: 43.9%-100% |
| Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 4 | 0 | 0 |
| C/T (heterozygous) | 0 | 1 | 0 |
| T/T (homozygous rare) | 0 | 0 | 3 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=8 | 4 | 1 | 3 |
| PA (C/C | C/C) = 100% (4/4) with 95% CI: 51.0%-100% |
| PA (C/T | C/T) = 100% (1/1) with 95% CI: 20.7%-100% |
| PA (T/T | T/T) =100% (3/3) with 95% CI: 43.9%-100% |
| Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 215 | 0 | 0 |
| C/T (heterozygous) | 0 | 6 | 0 |
| T/T (homozygous rare) | 0 | 0 | 6 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=227 | 215 | 6 | 6 |
| PA (C/C | C/C) = 100% (215/215) with 95% CI: 98.2%-100% |
| PA (C/T | C/T) = 100% (6/6) with 95% CI: 61.0%-100% |
| PA (T/T | T/T) = 100% (6/6) with 95% CI: 43.9%-100% |
| Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7% |
| Minor allele frequency for PI*M procida SERPINA1 in the 1000 Genomes Project was <0.001%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of C/T and T/T are 97.603% and 99.999% correspondingly |

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| PI* M malton variant (c.226_228delTTC) of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| TTC/TTC | TTC/delTTC | delTTC/delTTC |
| AlphaID At Home Genetic Health Risk Service | TTC/TTC (homozygous common) | 195 | 0 | 0 |
| TTC/ delTTC (heterozygous) | 0 | 21 | 0 |
| delTTC/ delTTC (homozygous rare) | 0 | 0 | 3 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=219 | 195 | 21 | 3 |
| PA (TTC/TTC | TTC/TTC) = 100% (195/195) with 95% CI: 98.1%-100% |
| PA (TTC/delTTC | TTC/delTTC) = 100% (21/21) with 95% CI: 84.5%-100% |
| PA (delTTC/delTTC | delTTC/delTTC) = 100% (3/3) with 95% CI: 43.9%-100% |
| Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| TTC/TTC | TTC/delTTC | delTTC/delTTC |
| AlphaID At Home Genetic Health Risk Service | TTC/TTC (homozygous common) | 4 | 0 | 0 |
| TTC/delTTC (heterozygous) | 0 | 1 | 0 |
| delTTC/delTTC (homozygous rare) | 0 | 0 | 3 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=8 | 4 | 1 | 3 |
| PA (TTC/TTC | TTC/TTC) = 100% (4/4) with 95% CI: 51.0%-100% |
| PA (TTC/delTTC | TTC/delTTC) = 100% (1/1) with 95% CI: 20.7%-100% |
| PA (delTTC/delTTC | delTTC/delTTC) =100% (3/3) with 95% CI: 43.9%-100% |
| Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| TTC/TTC | TTC/delTTC | delTTC/delTTC |
| AlphaID At Home Genetic Health Risk Service | TTC/TTC (homozygous common) | 199 | 0 | 0 |
| TTC/ delTTC (heterozygous) | 0 | 22 | 0 |
| delTTC/ delTTC (homozygous rare) | 0 | 0 | 6 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=227 | 199 | 22 | 6 |
| PA (TTC/TTC | TTC/TTC) = 100% (199/199) with 95% CI: 98.1%-100% |
| PA (TTC/delTTC | TTC/delTTC) = 100% (22/22) with 95% CI: 85.1%-100% |
| PA (delTTC/delTTC | delTTC/delTTC) = 100% (6/6) with 95% CI: 61.0%-100% |
| Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7% |
| Minor allele frequency for PI*M malton SERPINA1 in the 1000 Genomes Project was <0.001%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of TTC/delTTC and delTTC/delTTC are 99.382% and 99.996% correspondingly. |

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| PI* S iiyama variant (c.230C>T) of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 217 | 0 | 0 |
| C/T (heterozygous) | 0 | 2 | 0 |
| T/T (homozygous rare) | 0 | 0 | 0 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=219 | 217 | 2 | 0 |
| PA (C/C | C/C) = 100% (217/217) with 95% CI: 98.3%-100% PA (C/T | C/T) = 100% (2/2) with 95% CI: 34.2%-100% PA (T/T | T/T) = N/A Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 4 | 0 | 0 |
| C/T (heterozygous) | 0 | 1 | 0 |
| T/T (homozygous rare) | 0 | 0 | 3 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=8 | 4 | 1 | 3 |
| PA (C/C | C/C) = 100% (4/4) with 95% CI: 51.0%-100% PA (C/T | C/T) = 100% (1/1) with 95% CI: 20.7%-100% PA (T/T | T/T) = 100% (3/3) with 95% CI: 43.9%-100% Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 221 | 0 | 0 |
| C/T (heterozygous) | 0 | 3 | 0 |
| T/T (homozygous rare) | 0 | 0 | 3 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=227 | 221 | 3 | 3 |
| PA (C/C | C/C) = 100% (221/221) with 95% CI: 98.3%-100% PA (C/T | C/T) = 100% (3/3) with 95% CI: 43.9%-100% PA (T/T | T/T) = 100% (3/3) with 95% CI: 43.9%-100% Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7% Minor allele frequency for PI*S iiyama SERPINA1 in the 1000 Genomes Project was <0.001%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of C/T and T/T are 95.257% and 99.9995% correspondingly. |

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{14}

| PI*Q0 granite falls (c.552delC) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/delC | delC/delC |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 216 | 0 | 0 |
| C/ delC (heterozygous) | 0 | 3 | 0 |
| delC/delC (homozygous rare) | 0 | 0 | 0 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=219 | 216 | 3 | 0 |
| PA (C/C | C/C) = 100% (216/216) with 95% CI: 98.3%–100% PA (C/delC | C/delC) = 100% (3/3) with 95% CI: 43.9%–100% PA (delC/delC | delC/delC) = N/A Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%–1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/delC | delC/delC |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 0 | 0 | 0 |
| C/delC (heterozygous) | 0 | 2 | 0 |
| delC/delC (homozygous rare) | 0 | 0 | 6 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=8 | 0 | 2 | 6 |
| PA (C/C | C/C) = N/A PA (C/delC | C/delC) = 100% (2/2) with 95% CI: 34.2%–100% PA (delC/delC | delC/delC) =100% (6/6) with 95% CI: 61.0%–100% Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%–32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/delC | delC/delC |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 216 | 0 | 0 |
| C/ delC (heterozygous) | 0 | 5 | 0 |
| delC/delC (homozygous rare) | 0 | 0 | 6 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=227 | 216 | 5 | 6 |
| PA (C/C | C/C) = 100% (216/216) with 95% CI: 98.3%–100% PA (C/delC | C/delC) = 100% (5/5) with 95% CI: 56.6%–100% PA (delC/delC | delC/delC) = 100% (6/6) with 95% CI: 61.0%–100% Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%–1.7% Minor allele frequency for PI*Q0 granite falls SERPINA1 in the 1000 Genomes Project was <0.001%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of C/delC and delC/delC are 97.124 and 99.999% correspondingly. |

K221420 - Page 15 of 64

{15}

| PI*Q0 west (646+1G>T) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| G/G | G/T | T/T |
| AlphaID At Home Genetic Health Risk Service | G/G (homozygous common) | 215 | 0 | 0 |
| G/T (heterozygous) | 0 | 4 | 0 |
| T/T (homozygous rare) | 0 | 0 | 0 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=219 | 215 | 4 | 0 |
| PA (G/G | G/G) = 100% (215/215) with 95% CI: 98.2%-100% PA (G/T | G/T) = 100% (4/4) with 95% CI: 51.0%-100% PA (T/T | T/T) = N/A Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| G/G | G/T | T/T |
| AlphaID At Home Genetic Health Risk Service | G/G (homozygous common) | 0 | 0 | 0 |
| G/T (heterozygous) | 0 | 2 | 0 |
| T/T (homozygous rare) | 0 | 0 | 6 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=8 | 0 | 2 | 6 |
| PA (G/G | G/G) = N/A PA (G/T | G/T) = 100% (2/2) with 95% CI: 34.2%-100% PA (T/T | T/T) =100% (6/6) with 95% CI: 61.0%-100% Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| G/G | G/T | T/T |
| AlphaID At Home Genetic Health Risk Service | G/G (homozygous common) | 215 | 0 | 0 |
| G/T (heterozygous) | 0 | 6 | 0 |
| T/T (homozygous rare) | 0 | 0 | 6 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=227 | 215 | 6 | 6 |
| PA (G/G | G/G) = 100% (215/215) with 95% CI: 98.2%-100% PA (G/T | G/T) = 100% (6/6) with 95% CI: 61.0%-100% PA (T/T | T/T) = 100% (6/6) with 95% CI: 61.0% - 100%. Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7%. Minor allele frequency for PI*Q0 west SERPINA1 in the 1000 Genomes Project was <0.001%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of G/T and T/T are 97.603% and 99.999% correspondingly. |

K221420 - Page 16 of 64

{16}

| PI*Q0 bellingham (c.721A>T) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| A/A | A/T | T/T |
| AlphaID At Home Genetic Health Risk Service | A/A (homozygous common) | 213 | 0 | 0 |
| A/T (heterozygous) | 0 | 5 | 0 |
| TT (homozygous rare) | 0 | 0 | 1 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=219 | 213 | 5 | 1 |
| PA (A/A | A/A) = 100% (213/213) with 95% CI: 98.2%-100% PA (A/T | A/T) = 100% (5/5) with 95% CI: 56.6%-100% PA (T/T | T/T) = 100% (1/1) with 95% CI: 94.7%-100% Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| A/A | A/T | T/T |
| AlphaID At Home Genetic Health Risk Service | A/A (homozygous common) | 0 | 0 | 0 |
| A/T (heterozygous) | 0 | 2 | 0 |
| TT (homozygous rare) | 0 | 0 | 6 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=8 | 0 | 2 | 6 |
| PA (A/A | A/A) = N/A PA (A/T | A/T) = 100% (2/2) with 95% CI: 34.2%-100% PA (T/T | T/T) = 100% (6/6) with 95% CI: 61.0%-100% Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| A/A | A/T | T/T |
| AlphaID At Home Genetic Health Risk Service | A/A (homozygous common) | 213 | 0 | 0 |
| A/T (heterozygous) | 0 | 7 | 0 |
| TT (homozygous rare) | 0 | 0 | 7 |
| 'No call' or 'Invalid test'' | 0 | 0 | 0 |
| Total=227 | 213 | 7 | 7 |
| PA (A/A | A/A) = 100% (213/213) with 95% CI: 98.2%-100% PA (A/T | A/T) = 100% (7/7) with 95% CI: 64.6%-100% PA (T/T | T/T) = 100% (7/7) with 95% CI: 64.6%-100% Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7%. Minor allele frequency for PI*Q0 bellingham SERPINA1 in the 1000 Genomes Project was <0.001%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of A/T and T/T are 97.947% and 99.999% correspondingly. |

K221420 - Page 17 of 64

{17}

| PI*F (c.739C>T) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 194 | 0 | 0 |
| C/T (heterozygous) | 0 | 23 | 0 |
| T/T (homozygous rare) | 0 | 0 | 2 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=219 | 194 | 23 | 2 |
| PA (C/C | C/C) = 100% (194/194) with 95% CI: 98.1%-100% PA (C/T | C/T) = 100% (23/23) with 95% CI: 85.7%-100% PA (T/T | T/T) = 100% (2/2) with 95% CI: 34.2%-100% Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 0 | 0 | 0 |
| C/T (heterozygous) | 0 | 2 | 0 |
| T/T (homozygous rare) | 0 | 0 | 6 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=8 | 0 | 2 | 6 |
| PA (C/C | C/C) = N/A PA (C/T | C/T) = 100% (2/2) with 95% CI: 34.2%-100% PA (T/T | T/T) = 100% (6/6) with 95% CI: 61.0%-100% Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 194 | 0 | 0 |
| C/T (heterozygous) | 0 | 25 | 0 |
| T/T (homozygous rare) | 0 | 0 | 8 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=227 | 194 | 25 | 8 |
| PA (C/C | C/C) = 100% (194/194) with 95% CI: 98.1%-100% PA (C/T | C/T) = 100% (25/25) with 95% CI: 86.7%-100% PA (T/T | T/T) = 100% (8/8) with 95% CI: 67.6%-100% Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7% Minor allele frequency for PI*F SERPINA1 in the 1000 Genomes Project was 0.1%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of C/T and T/T are 99.464% and 99.996% correspondingly. |

K221420 - Page 18 of 64

{18}

| PI*P lowell (c.839A>T) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| A/A | A/T | T/T |
| AlphaID At Home Genetic Health Risk Service | A/A (homozygous common) | 201 | 0 | 0 |
| A/T (heterozygous) | 0 | 14 | 0 |
| TT (homozygous rare) | 0 | 0 | 4 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=219 | 201 | 14 | 4 |
| PA (A/A | A/A) = 100% (201/201) with 95% CI: 98.1%-100% PA (A/T | A/T) = 100% (14/14) with 95% CI: 78.5%-100% PA (T/T | T/T) = 100% (4/4) with 95% CI: 51.0%-100% Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| A/A | A/T | T/T |
| AlphaID At Home Genetic Health Risk Service | A/A (homozygous common) | 0 | 0 | 0 |
| A/T (heterozygous) | 0 | 2 | 0 |
| TT (homozygous rare) | 0 | 0 | 6 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=8 | 0 | 2 | 6 |
| PA (A/A | A/A) = N/A PA (A/T | A/T) = 100% (2/2) with 95% CI: 34.2%-100% PA (T/T | T/T) =100% (6/6) with 95% CI: 61.0%-100% Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4%. |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| A/A | A/T | T/T |
| AlphaID At Home Genetic Health Risk Service | A/A (homozygous common) | 201 | 0 | 0 |
| A/T (heterozygous) | 0 | 16 | 0 |
| TT (homozygous rare) | 0 | 0 | 10 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=227 | 201 | 16 | 10 |
| PA (A/A | A/A) = 100% (201/201) with 95% CI: 98.1%-100% PA (A/T | A/T) = 100% (16/16) with 95% CI: 80.6%-100% PA (T/T | T/T) = 100% (10/10) with 95% CI: 72.2%-100% Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7% Minor allele frequency for PI*P lowell SERPINA1 in the 1000 Genomes Project was <0.001%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of A/T and T/T are 99.464% and 99.996% correspondingly. |

K221420 - Page 19 of 64

{19}

| PI*S (c.863A>T) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| A/A | A/T | T/T |
| AlphaID At Home Genetic Health Risk Service | A/A (homozygous common) | 163 | 0 | 0 |
| A/T (heterozygous) | 0 | 36 | 0 |
| T/T (homozygous rare) | 0 | 0 | 20 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=219 | 163 | 36 | 20 |
| PA (A/A | A/A) = 100% (163/163) with 95% CI: 97.7%-100% |
| PA (A/T | A/T) = 100% (36/36) with 95% CI: 90.4%-100% |
| PA (T/T | T/T) = 100% (20/20) with 95% CI: 83.9%-100% |
| Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| A/A | A/T | T/T |
| AlphaID At Home Genetic Health Risk Service | A/A (homozygous common) | 0 | 0 | 0 |
| A/T (heterozygous) | 0 | 2 | 0 |
| T/T (homozygous rare) | 0 | 0 | 6 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=8 | 0 | 2 | 6 |
| PA (A/A | A/A) = N/A |
| PA (A/T | A/T) = 100% (2/2) with 95% CI: 34.2%-100% |
| PA (T/T | T/T) = 100% (6/6) with 95% CI: 61.0%-100% |
| Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| A/A | A/T | T/T |
| AlphaID At Home Genetic Health Risk Service | A/A (homozygous common) | 163 | 0 | 0 |
| A/T (heterozygous) | 0 | 38 | 0 |
| T/T (homozygous rare) | 0 | 0 | 26 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=227 | 163 | 38 | 26 |
| PA (A/A | A/A) = 100% (163/163) with 95% CI: 97.7%-100% |
| PA (A/T | A/T) = 100% (38/38) with 95% CI: 90.8%-100% |
| PA (T/T | T/T) = 100% (26/26) with 95% CI: 87.1%-100% |
| Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7%. |
| Minor allele frequency for PI*S SERPINA1 in the 1000 Genomes Project was 2%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of A/T and T/T are 99.669% and 99.992% correspondingly. |

K221420 - Page 20 of 64

{20}

| PI*Z (c.1096G>A) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| G/G | G/A | A/A |
| AlphaID At Home Genetic Health Risk Service | G/G (homozygous common) | 167 | 0 | 0 |
| G/A (heterozygous) | 0 | 37 | 0 |
| A/A (homozygous rare) | 0 | 0 | 15 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=219 | 167 | 37 | 15 |
| PA (G/G | GG) = 100% (167/167) with 95% CI: 97.8%-100% PA (G/A | G/A) = 100% (37/37) with 95% CI: 90.6%-100% PA (A/A | A/A) = 100% (15/15) with 95% CI: 79.6%-100% Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| G/G | G/A | A/A |
| AlphaID At Home Genetic Health Risk Service | G/G (homozygous common) | 0 | 0 | 0 |
| G/A (heterozygous) | 0 | 2 | 0 |
| A/A (homozygous rare) | 0 | 0 | 6 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=8 | 0 | 2 | 6 |
| PA (G/G | G/G) = N/A PA (G/A | G/A) = 100% (2/2) with 95% CI: 34.2%-100% PA (A/A | A/A) = 100% (6/6) with 95% CI: 61.0%-100% Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4%. |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| G/G | G/A | A/A |
| AlphaID At Home Genetic Health Risk Service | G/G (homozygous common) | 167 | 0 | 0 |
| G/A (heterozygous) | 0 | 39 | 0 |
| A/A (homozygous rare) | 0 | 0 | 21 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=227 | 167 | 39 | 21 |
| PA(G/G | GG) = 100% (167/167) with 95% CI: 97.8%-100% PA (G/A | G/A) = 100% (39/39) with 95% CI: 91.0%-100% PA (A/A | A/A) = 100% (21/21) with 95% CI: 84.5%-100% Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7%. Minor allele frequency for PI*Z SERPINA1 in the 1000 Genomes Project was 0.4%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of G/A and A/A are 99.680% and 99.992% correspondingly |

K221420 - Page 21 of 64

{21}

| PI*Q0 mattawa (c.1130dupT) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| T/T | T/dupT | dupT/dupT |
| AlphaID At Home Genetic Health Risk Service | T/T (homozygous common) | 212 | 0 | 0 |
| T/dupT (heterozygous) | 0 | 5 | 0 |
| dupT/dupT (homozygous rare) | 0 | 0 | 2 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=219 | 212 | 5 | 2 |
| PA (T/T | T/T) = 100% (212/212) with 95% CI: 98.2%-100% PA (T/dupT | T/dupT) = 100% (5/5) with 95% CI: 56.6%-100% PA (dupT/dupT | dupT/dupT) = 100% (2/2) with 95% CI: 34.2%-100% Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| T/T | T/dupT | dupT/dupT |
| AlphaID At Home Genetic Health Risk Service | T/T (homozygous common) | 0 | 0 | 0 |
| T/dupT (heterozygous) | 0 | 2 | 0 |
| dupT/dupT (homozygous rare) | 0 | 0 | 6 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=8 | 0 | 2 | 6 |
| PA (T/T | T/T) = N/A PA (T/dupT | T/dupT) = 100% (2/2) with 95% CI: 34.2%-100% PA (dupT/dupT | dupT/dupT) = 100% (6/6) with 95% CI: 61.0%-100% Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| T/T | T/dupT | dupT/dupT |
| AlphaID At Home Genetic Health Risk Service | T/T (homozygous common) | 212 | 0 | 0 |
| T/dupT (heterozygous) | 0 | 7 | 0 |
| dupT/dupT (homozygous rare) | 0 | 0 | 8 |
| ‘No call’ or ‘Invalid test’ | 0 | 0 | 0 |
| Total=227 | 212 | 7 | 8 |
| PA (T/T | T/T) = 100% (212/212) with 95% CI: 98.2%-100% PA (T/dupT | T/dupT) = 100% (7/7) with 95% CI: 64.6%-100% PA (dupT/dupT | dupT/dupT) = 100% (8/8) with 95% CI: 67.6%-100% Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7% Minor allele frequency for PI*Q0 mattawa SERPINA1 in the 1000 Genomes Project was <0.001%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of T/dupT and dupT/dupT are 97.947% and 99.999% correspondingly. |

K221420 - Page 22 of 64

{22}

| PI*Q0 clayton (c.1158dupC) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| CC | C/dupC | dupC/dupC |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 217 | 0 | 0 |
| C/dupC (heterozygous) | 0 | 2 | 0 |
| dupC/dupC (homozygous rare) | 0 | 0 | 0 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=219 | 217 | 2 | 0 |
| PA (C/C | C/C) = 100% (217/217) with 95% CI: 98.3%–100% PA (C/dupC | C/dupC) = 100% (2/2) with 95% CI: 34.2%–100% PA (dupC/dupC | dupC/dupC) = N/A Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%–1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| CC | C/dupC | dupC/dupC |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 0 | 0 | 0 |
| C/dupC (heterozygous) | 0 | 2 | 0 |
| dupC/dupC (homozygous rare) | 0 | 0 | 6 |
| 'No call' or 'Invalid test'' | 0 | 0 | 0 |
| Total=8 | 0 | 2 | 6 |
| PA (C/C | C/C) = N/A PA (C/dupC | C/dupC) = 100% (2/2) with 95% CI: 34.2%–100% PA (dupC/dupC | dupC/dupC) =100% (6/6) with 95% CI: 61.0%–100% Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%–32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| CC | C/dupC | dupC/dupC |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 217 | 0 | 0 |
| C/dupC (heterozygous) | 0 | 4 | 0 |
| dupC/dupC (homozygous rare) | 0 | 0 | 6 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=227 | 217 | 4 | 6 |
| PA (C/C | C/C) = 100% (217/217) with 95% CI: 98.3%–100% PA (C/dupC | C/dupC) = 100% (4/4) with 95% CI: 51.0%–100% PA (dupC/dupC | dupC/dupC) = 100% (6/6) with 95% CI: 61.0%–100% Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%–1.7% Minor allele frequency for PI*Q0 clayton SERPINA1 in the 1000 Genomes Project was <0.001%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of C/dupC and dupC/dupC are 96.415% and 99.999% correspondingly. |

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| PI*M heerlen (c.1178C>T) variant of the SERPINA1 gene |
| --- |
| Clinical Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 210 | 0 | 0 |
| C/T (heterozygous) | 0 | 5 | 0 |
| T/T (homozygous rare) | 0 | 0 | 4 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=219 | 210 | 5 | 4 |
| PA (C/C | C/C) = 100% (210/210) with 95% CI: 98.2%-100% |
| PA (C/T | C/T) = 100% (5/5) with 95% CI: 56.6%-100% |
| PA (T/T | T/T) = 100% (4/4) with 95% CI: 51.0%-100% |
| Percent of no calls or invalid is 0.0% (0/219) with 95% CI: 0.0%-1.7% |
| Contrived Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 0 | 0 | 0 |
| C/T (heterozygous) | 0 | 2 | 0 |
| T/T (homozygous rare) | 0 | 0 | 6 |
| 'no calls' or 'invalid' | 0 | 0 | 0 |
| Total=8 | 0 | 2 | 6 |
| PA (C/C | C/C) = N/A |
| PA (C/T | C/T) = 100% (2/2) with 95% CI: 34.2%-100% |
| PA (T/T | T/T) = 100% (6/6) with 95% CI: 61.0%-100% |
| Percent of no calls or invalid is 0.0% (0/8) with 95% CI: 0.0%-32.4% |
| All Samples | Genotype | Sanger Bidirectional Sequencing |
| C/C | C/T | T/T |
| AlphaID At Home Genetic Health Risk Service | C/C (homozygous common) | 210 | 0 | 0 |
| C/T (heterozygous) | 0 | 7 | 0 |
| T/T (homozygous rare) | 0 | 0 | 10 |
| 'No call' or 'Invalid test' | 0 | 0 | 0 |
| Total=227 | 210 | 7 | 10 |
| PA (C/C | C/C) = 100% (210/210) with 95% CI: 98.2%-100% |
| PA (C/T | C/T) = 100% (7/7) with 95% CI: 64.6%-100% |
| PA (T/T | T/T) = 100% (10/10) with 95% CI: 72.2%-100% |
| Percent of no calls or invalid is 0.0% (0/227) with 95% CI: 0.0%-1.7% |
| Minor allele frequency for PI*M heerlen SERPINA1 in the 1000 Genomes Project was <0.001%; technical (analytical) positive predictive values for AlphaID At Home Genetic Health Risk Service test result of C/T and T/T are 97.947% and 99.999% correspondingly. |

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2. Matrix Comparison:

Not applicable

# C Clinical Studies:

1. Clinical Sensitivity:

Not applicable

2. Clinical Specificity:

Not applicable

3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):

a) Risk of developing lung and liver disease associated with AATD depends on specific variant combination detected

AATD is a genetic autosomal recessive disease with codominant expression (i.e., each of the two alleles present in each person contributes 50% on the total generated AAT protein), characterized by low levels of the AAT protein in the blood, that may lead to various health conditions. The most common diseases are lung and/or liver disease. AATD occurs in people of all ethnicities worldwide.¹ However, it is most common in people of European descent. AATD affects about 1 in 1,500 to 3,500 people of European descent.²,³ Many individuals with AATD are likely undiagnosed, particularly people with a lung condition called chronic obstructive pulmonary disease (COPD). COPD can be caused by AATD; however, AATD is rarely diagnosed. Some people with AATD are misdiagnosed with asthma.⁴ Two to three percent of patients with COPD in the United States are estimated to have AATD.² The percentage of patients with liver disease who have AATD is not known.

AATD is caused by modifications in the genetic sequence of the SERPINA1 gene, which give rise to different variants. More than 120 variants in the SERPINA1 gene have been identified. Some of these variants do not affect the production of AAT protein, but some others may differently affect the levels or functionality of the AAT protein and, therefore, each specific variant may be associated with different probabilities for the development of lung disease and/or liver disease.

The AlphaID At Home Genetic Health Risk Service is indicated for the detection of

¹ Stoller JK, Hupertz V, Aboussouan LS. Alpha-1 Antitrypsin Deficiency. Deficiency. 2006 Oct 27 [Updated 2020 May 21]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2020

² American Thoracic Society; European Respiratory Society. American Thoracic Society/European Respiratory Society statement: standards for the diagnosis and management of individuals with alpha-1 antitrypsin deficiency. Am J Respir Crit Care Med. 2003 Oct 1;168(7):818-900

³ de Serres F, Blanco I. Role of alpha-1 antitrypsin in human health and disease. J Intern Med.2014 Oct;276(4):311-35.

⁴ Genetics Home Reference-NIH (https://ghr.nlm.nih.gov/condition/alpha-1-antitrypsindeficiency# statistics)

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14 variants in the SERPINA1 gene associated with AATD. These 14 variants explain 95% of AATD cases¹ and are mainly found in European population, except for PI*S iiyama, which was described in the Asian population.² The most common variants are PI*S and PI*Z. Published studies estimate frequency ranges of 5–10% and 1–3% for PI*S and PI*Z, respectively, in the European population.³ An analysis of the prevalence of PI*S and PI*Z amongst the five major ethnic subgroups in the United States has demonstrated that the highest risk for AATD is found in Caucasians, followed by Hispanics and Blacks, with the lowest prevalence amongst Mexican Americans and no risk amongst Asian.³ The remaining 12 genetic variants tested are reported with a very low frequency in the population.³

Considering that a given person can have a genetic result of zero, one or two copies of the variants detected, a total of 120 possible genotype combinations of these 14 variants can be reported by the AlphaID At Home Genetic Health Risk Service:

- 1 genetic result with 0 variants detected (wild-type homozygous),
- 14 genetic results with 1 variant detected (heterozygous), and
- 105 genetic results with 2 variants detected (rare homozygous or compound heterozygous).

In some rare circumstances, a “variant not determined” result could be reported, which means that the Service cannot determine if the user has any of the 14 tested variants in the SERPINA1 gene linked to AATD. This is an inconclusive genetic result. The presence of any other rare genetic variant in the DNA sequence can interfere with the genetic analysis, leading to the inability to determine the genetic result. In this case, the risk of developing lung and liver disease cannot be determined, and a different test/Service is recommended to the user (DNA sequencing of the SERPINA1 gene).

Depending on the specific variant combination detected, the AlphaID At Home Genetic Health Risk Service provides the individuals’ genetic health risk for developing lung and/or liver disease linked to AATD. These variants can cause mild, severe, or very severe plasma deficiency and can cause no accumulation or mild or severe accumulation of protein in the liver. Normal AAT levels (80–120%) are defined by serum AAT levels above 20 μmol/L (measured by nephelometry).³ It is considered that severe AATD is defined by serum AAT levels below the lung protective threshold of 35% of the mean expected value (50 mg/dl or 11 μmol/L measured by nephelometry, or 80 mg/dL measured by radial immunodiffusion).²,³ Very severe AATD is defined by no active AAT or AAT levels below 1%. AAT levels for the most frequent and most studies studied genetic results are reported in Stoller et al. 2017¹ and provided in the table below.

⁵ Seyama K. State of alpha1-antitrypsin deficiency in Japan. Respirology. 2001 Jun;6 Suppl:S35-8.

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| AAT Protein Variants | Serum AAT Levels | | AAT Protein Reduction |
| --- | --- | --- | --- |
| | True Level Mean, μmol/L (5^{th}–95^{th} percentile) | Commercial Standard Median, mg/dL (5^{th}–95^{th} percentile) | |
| PI*M/PI*M | 33 (20–53) | 147 (102–254) | Normal-Mild |
| PI*M/PI*S | 33 (18–52) | 125 (86–218) | |
| PI*M/PI*Z | 25.4 (15–42) | 90 (62–151) | |
| PI*S/PI*S | 28 (20–48) | 95 (43–154) | |
| PI*S/PI*Z | 16.5 (10–23) | 62 (233–108) | Mild severe |
| PI*Z/PI*Z | 5.3 (3.4–7) | ≤29 (≤29–52) | Severe |
| Null-Null* | 0 | 0 | Very severe |
| *Null variants include PI*Q0 granite falls, PI*Q0 west, PI*Q0 bellingham, PI*Q0 mattawa and PI*Q0 clayton | | | |

The risk categorization is based on the reported clinical cases (published references) for each genetic result. If any of the 14 variants tested is detected, one of the four following risk categories will be reported:

1) Increased risk: There is an increased risk of developing lung or liver disease linked to AATD compared to the general population. The chance of developing lung or liver disease linked to AATD is higher than that of the general population. Above 80% of people with this genetic result develop lung or liver disease during their lifetime.
2) Slightly at Increased risk: There is a slightly increased risk of developing lung or liver disease linked to AATD compared to the general population. The chance of developing lung or liver disease linked to AATD is slightly higher than that of the general population. 20–80% of people with this genetic result develop lung or liver disease during their lifetime.
3) Not likely at increased risk: There is average risk of developing lung or liver disease linked to AATD compared to the general population. The chance of developing lung or liver disease linked to AATD is similar to that of the general population. Below 20% of people with this genetic result develop lung or liver disease during their lifetime.
4) Unknown risk: The risk of developing lung or liver disease linked to AATD is not known due to the lack of reported clinical cases or inconclusive data. The chance of developing lung or liver disease linked to AATD is unknown. More clinical studies are needed to determine the risk level.

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When No Variants are detected by this Service, the result will show ‘*Not Likely at Risk for AATD*’. It cannot rule out the possibility of having an extremely rare variant linked to AATD not tested by this Service. It is still possible to have a higher risk category of developing lung and/or liver disease linked to AATD.

These reported risk categories are based on the evidence in the scientific literature and are summarized below separately for lung disease and liver disease:

# *a. Lung disease risk categorization*

The AAT protein is made in the liver, released into the bloodstream, and enters the lungs, where it inactivates neutrophil elastase, thereby protecting the lung tissue from protease mediated damage. As such, the plasma levels of this protein have widely been described to be directly related to the amount that is reaching the lungs, and consequently, with its lung protection capability. 2% to 3% of patients with COPD in the United States are estimated to have AATD.$^{2}$ Symptomatic obstructive lung disease in AATD usually presents in patients in their 30s and 40s. Considerable variability in the time of onset of symptoms has been described. Although severe symptoms are most often seen in current or previous cigarette smokers, some smokers and many nonsmokers develop no symptoms at all.$^{2,3,6,7}$

The classic pulmonary presentation of AATD is a severe, early onset panacinar emphysema with a basilar predominance in adults. However, emphysema may also occur in a diffuse distribution or predominantly in the upper lobes.$^{2,3,6,7}$ In addition to emphysema, AATD is also characterized by chronic, progressive fatigue and other nonspecific respiratory symptoms.$^{3}$ Shortness of breath, medically known as dyspnea, is generally the prominent symptom, but chronic cough or wheezing may also occur, as early as age 18 years.$^{2,6}$ Pulmonary function testing show findings consistent with COPD; however, bronchodilator responsiveness may be seen and may be labelled as asthma.$^{7}$ The presence of episodic wheezing and dyspnea consistent with a diagnosis of asthma has been noted in AATD.$^{2}$ Recurring lung infections are also shown in subjects with AATD.$^{2}$ Bronchiectasis, with or without concomitant emphysema, is less common.$^{6,7}$ Although it is likely that AATD can promote the development of bronchial asthma and bronchiectasis, there is no reliable evidence as to whether AATD influences the frequency or gravity of these diseases.$^{3}$

The reported lung disease risk category has been determined based on the clinical outcome which includes chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis, and bronchiectasis of the reported cases for each genetic result. The following general criteria summarized in the table below has been followed for the assignment of a risk category for each genetic result. The “*Unknown risk*” category is assigned to genotypes with <3 reported clinical cases, as well those with ≥3 reported clinical cases but with a calculated risk confidence interval overlapping with the other two risk categories.

$^{6}$ Silverman EK, Sandhaus RA. Clinical practice. Alpha1-antitrypsin deficiency. N Engl J Med. 2009 Jun 25;360(26):2749-57. doi: 10.1056/NEJMcp0900449. Review. PubMed PMID: 19553648.

$^{7}$ Brode SK, Ling SC, Chapman KR. Alpha-1 antitrypsin deficiency: a commonly overlooked cause of lung disease. CMAJ. 2012 Sep 4;184(12):1365-71. doi: 10.1503/cmaj.111749.

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| General Criteria for Lung Disease Risk Categorization | | | |
| --- | --- | --- | --- |
| Number of reported clinical cases | %Reported clinical cases with lung disease | 95% CI | Reported lung disease risk category* |
| n<3 | Any | Any | Unknown risk |
| n≥3 | <20% | 0–80% | Not Likely at increased risk |
| | | 0–100% | Unknown risk |
| | 20-80% | 0–80% | Not Likely at increased risk |
| | | 20–100% | Slightly increased risk |
| | | 0–100% | Unknown risk |
| | >80% | 0–100% | Unknown risk |
| | | 20–100% | Slightly increased risk |
| | | >80% | Increased risk |

NOTE: Exceptions to this general classification are:

• All carriers with at least one PI*M allele (except PI*M/PI*Z ever-smokers) will be reported as 'Not likely at increased risk'

• PI*Z/PI*Null and PI*Null/PI*Null combinations will be assigned as 'Increased risk'. Null variants: PI*Q0 granite falls, PI*Q0 west, PI*Q0 bellingham, PI*Q0 mattawa and PI*Q0 clayton

* The reported risk category may be updated if there is enough data to provide confidence intervals that do not overlap between two consecutive risk categories

The lung disease risk categorization along with the 95% confidence intervals (CIs) of the risk estimates for the 119 genetic results with one or two variants detected are presented in the table below based on the number of reported clinical cases extracted from the referenced scientific literature and meta-analyses that analyze clinical cases with and without lung disease. Pending new clinical cases being published, the risk category is to be updated if there is enough data to provide confidence intervals that do not overlap between two consecutive risk categories. Since the intended population of the Service is ≥18 years of age, only cases (≥18 years old) have been included in the analysis to estimate the risk, when the age date was described in the study. The exceptions to the general criteria for liver disease risk categorization are shaded in grey in the table below.

| # | Lung Disease Risk Categorization | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | Genetic Results | Reported Clinical Cases with Lung Disease | Reported Clinical Cases without Lung Disease | %Reported Clinical Cases with Lung Disease^{1} | 95% CI^{1} | Reported Lung Disease Risk Category |
| **Most frequent^{2} genetic results (#1– #6)** | | | | | | |
| 1 | **PI*M/PI*M** | N/A | N/A | N/A | N/A | **Not Likely at increased risk** |
| | None of the 14 allelic variants interrogated by the A1AT Genotyping test are detected in the SERPINA1 gene, but other variants could be present | | | | | |
| | **PI*M/** | | | | | |

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| 2 | **PI*Z** | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | 155 | 1967 | 7.3 | 6.4 – 8.3 | **Not Likely at increased risk** |
| | Never-smokers | Dahl *et al.*, 2002: 86 individuals with lung disease and 364 individuals without lung disease; Seersholm *et al.*, 2000: 47 individuals with lung disease and 1504 without lung disease; Klayton *et al.*, 1975: 13 individuals with lung disease and 14 without lung disease; Matzen *et al.*, 1977: 2 individuals with lung disease and 10 without lung disease; Gulsvik *et al.*, 1979: 7 individuals with lung disease and 44 without lung disease; Chang-Yeung *et al.*, 1978: 0 individuals with lung disease and 31 without lung disease. | | | | |
| | | 113 | 142 | 44.3 | 39.3 – 49.5 | **Slightly increased risk** |
| | Ever-smokers | Sørheim *et al.*, 2010: 43 individuals with lung disease and 72 without lung disease Molloy *et al.*, 2014: 64 individuals with lung disease and 78 without lung disease | | | | |
| | | 101 | 590 | 14.6 | 12.5 – 17.0 | **Not Likely at increased risk** |
| 3 | **PI*M/ PI*S** | Dahl *et al.*, 2002: 73 individuals with lung disease and 383 without lung disease Gulsvik *et al.*, 1979: 14 individuals with lung disease and 46 without lung disease Chang-Yeung *et al.*, 1978: 7 individuals with lung disease and 82 without lung disease Matzen *et al.*, 1977: 6 individuals with lung disease and 54 without lung disease Ostrow *et al.*, 1978: 1 individual with lung disease and 25 without lung disease | | | | |
| | | 4 | 42 | 8.7 | 4.0 – 18.0 | **Not Likely at increased risk** |
| 4 | **PI*S/ PI*S** | Sandford *et al.*, 1999 2 individuals with lung disease and 0 without lung disease; Gulsvik *et al.*, 1979 1 individual with lung disease and 0 without lung disease; Lieberman *et al.*, 1986 1 individual with lung disease and 2 without lung disease; Arnaud *et al.*, 1977 23 Individuals without lung disease; Dahl *et al.*, 2002 0 individuals with lung disease and 12 without lung disease; Fagerhol *et al.*, 1969 0 individuals with lung disease and 3 s without lung disease; Kueppers *et al.*, 1977 0 individuals with lung disease and 1 without lung disease; Lochon *et al.*, 1978 0 individuals with lung disease and 1 without lung disease | | | | |
| | | 269 | 228 | 54.1 | 50.1 – 57.8 | **Slightly increased risk** |
| 5 | **PI*S/ PI*Z** | McElvaney *et al.*, 2020: 269 individuals with lung disease and 181 without lung disease Bartmann *et al.*, 1985: 18 individuals with lung disease and 1 without lung disease Dahl *et al.*, 2002: 4 individuals with lung disease and 10 without lung disease Lieberman *et al.*, 1986: 2 individuals with lung disease and 5 without lung disease Abboud *et al.*, 1979: 1 individual with lung disease; Fagerhol *et al.*, 1969: 1 individual with lung disease and 1 individual without lung disease; Gulsvik *et al.*, 1979: 1 individual with lung disease and 3 individuals without lung disease | | | | |
| | | 2775 | 630 | 81.5 | 80.4 – 82.6 | **Increased risk** |
| 6 | **PI*Z /PI*Z** | McElvaney *et al.*, 2020: 2775 individuals with lung disease and 630 without lung disease | | | | |
| | | **Less common genetic results (#7–#120)** | | | | |
| 7 | **PI*S/ PI*I** | 0 | 2 | 0.0 | 0.0 – 57.5 | **Unknown risk** |
| | | Seri *et al.*, 1992 1 individual without lung disease; Huang *et al.*, 2017 1 individual without lung disease | | | | |
| 8 | **PI*F/ PI*S** | 0 | 1 | 0.0 | 0.0 – 73.0 | **Unknown risk** |
| | | Cook *et al.*, 1996 1 individual without lung disease | | | | |

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| 9 | **PI*I/ PI*I** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| --- | --- | --- | --- | --- | --- | --- |
| Clinical cases not reported | | | | | | |
| 10 | **PI*F/ PI*I** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 11 | **PI*F/ PI*F** | 1 | 0 | 100.0 | 27.0 – 100.0 | **Unknown risk** |
| Sinden *et al.*, 2014: 1 individual with lung disease | | | | | | |
| 12 | **PI*Z/ PI*I** | 4 | 0 | 100.0 | 59.6 – 95.5 | **Unknown risk** |
| Ferrarotti *et al.*, 2005: 2 individuals with lung disease Baur & Bencze 1987: 1 individual with lung disease Corda *et al.*, 2006: 1 individual with lung disease | | | | | | |
| 13 | **PI*F/ PI*Z** | 13 | 2 | 86.7 | 27.0 – 100.0 | **Slightly at increased risk** |
| Sinden *et al.*, 2014: 5 individuals with lung disease and 1 without lung disease; Cockcroft *et al.*, 1981: 3 individuals with lung disease; Cook *et al.*, 1996: 2 individuals with lung disease; Beckman *et al.*, 1984: 1 individual with lung disease; Franciosi *et al.*, 2019: 1 individual with lung disease; Kelly *et al.*, 1989: 1 individual with lung disease | | | | | | |
| 14 | **PI*S/ PI*M procida** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 15 | **PI*S/ PI*M malton** | 1 | 0 | 100 | 27.0 – 100.0 | **Unknown risk** |
| Figueira Gonçalves *et al.*, 2017: 1 individual with lung disease | | | | | | |
| 16 | **PI*S/ PI*iyama** | 0 | 0 | 0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 17 | **PI*S/ PI*P lowell** | 1 | 1 | 50.0 | 12.1 – 87.9 | **Unknown risk** |
| Jardi *et al.*, 1997: 1 individual without lung disease; Balbi *et al.*, 2019: 1 individual with lung disease | | | | | | |
| 18 | **PI*S/ PI*M heerlen** | 1 | 1 | 50.0 | 12.1 – 87.9 | **Unknown risk** |
| Kramps *et al.*, 1981: 1 individual with lung disease and 1 without lung disease | | | | | | |
| 19 | **PI*I/ PI*M procida** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 20 | **PI*I/ PI*M malton** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 21 | **PI*I/ PI*iyama** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 22 | **PI*M/ PI*P lowell** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 23 | **PI*I/ PI*M heerlen** | 0 | 0 | 0.0 | N/A | **Unknown risk** |

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| | Clinical cases not reported | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| 24 | **PI*F/ PI*M procida** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 25 | **PI*F/ PI*M malton** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 26 | **PI*F/ PI*iyama** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 27 | **PI*F/ PI*P lowell** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 28 | **PI*F/ PI*M heerlen** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 29 | **PI*S/ PI*Q0 granite falls** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 30 | **PI*S/ PI*Q0 west** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 31 | **PI*S/ PI*Q0 bellingham** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 32 | **PI*S/ PI*Q0 mattawa** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 33 | **PI*S/ PI*Q0 clayton** | 1 | 1 | 50.0 | 12.1 – 87.9 | **Unknown risk** |
| | Rosenbaum *et al.*, 2017: 1 individual with lung disease and 1 without lung disease | | | | | |
| 34 | **PI*I/ PI*Q0 granite falls** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 35 | **PI*I/ PI*Q0 west** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 36 | **PI*I/ PI*Q0 bellingham** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 37 | **PI*I/ PI*Q0 mattawa** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 38 | **PI*I/ PI*Q0 clayton** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |
| 39 | **PI*F/ PI*Q0 granite falls** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases not reported | | | | | |

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| 40 | **PI*F/ PI*Q0 west** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| --- | --- | --- | --- | --- | --- | --- |
| Clinical cases not reported | | | | | | |
| 41 | **PI*F/ PI*Q0 bellingham** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 42 | **PI*F/ PI*Q0 mattawa** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Cases not reported | | | | | | |
| 43 | **PI*F/ PI*Q0 clayton** | 1 | 0 | 100.0 | 27.0–100.0 | **Unknown risk** |
| Ringenbach *et al.*, 2011: 1 individual with lung disease | | | | | | |
| 44 | **PI*Z/ PI*M procida** | 2 | 0 | 100.0 | 42.5–100.0 | **Unknown risk** |
| Ferrarotti *et al.*, 2005: 1 individual with lung disease Lonardo *et al.*, 2002: 1 individual with lung disease | | | | | | |
| 45 | **PI*Z/ PI*M malton** | 13 | 0 | 100.0 | 82.5 – 100.0 | **Increased risk** |
| Sproule *et al.*, 1983: 4 individuals with lung disease; Ferrarotti *et al.*, 2005: 3 individuals with lung disease; Joly *et al.*, 2015: 2 individuals with lung disease; Allen *et al.*, 1986: 1 individual with lung disease; Corda *et al.*, 2006: 1 individual with lung disease; Suh-Lailam *et al.*, 2014: 1 individual with lung disease; Figueira Gonçalves *et al.*, 2017: 1 individual with lung disease | | | | | | |
| 46 | **PI*Z/ PI*iiyama** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 47 | **PI*Z/ PI*P lowell** | 7 | 0 | 100.0 | 72.1 – 100.0 | **Slightly at increased risk** |
| Bamforth & Kalsheker 1988: 4 individuals with lung disease; Esteves-Brandão *et al.*, 2019: 1 individual with lung disease; Holmes *et al.*, 1990: 1 individual with lung disease; Ferrarotti *et al.*, 2005: 1 with lung disease | | | | | | |
| 48 | **PI*Z/ PI*M heerlen** | 2 | 0 | 100.0 | 42.5 – 100.0 | **Unknown risk** |
| Klaassen *et al.*, 2001: 2 individuals with lung disease | | | | | | |
| 49 | **PI*M procida/ PI*M procida** | 2 | 0 | 100.0 | 42.5 – 100.0 | **Unknown risk** |
| Ferrarotti *et al.*, 2005: 2 individuals with lung disease | | | | | | |
| 50 | **PI*M malton/ PI*M procida** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 51 | **PI*M procida/ PI*iiyama** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Clinical cases not reported | | | | | | |
| 52 | **PI*M procida/ PI*P lowell** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Cases not reported | | | | | | |
| 53 | **PI*M procida/ PI*M heerlen** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| Cases not reported | | | | | | |
| 54 | **PI*M malton/ PI*M malton** | 19 | 5 | 79.2 | 63.0 – 89.5 | **Slightly at increased risk** |

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| | Orrù *et al.*, 2005: 7 individuals with lung disease and 3 without lung disease; Ferrarotti *et al.*, 2005: 5 individuals with lung disease; Reid *et al.*, 1987: 2 individuals with lung disease and 1 without lung disease; Figueira Gonçalves *et al.*, 2017: 1 individual with lung disease and 1 without lung disease; Curiel *et al.*, 1989: 1 individual with lung disease; Balduyck *et al.*, 2014: 1 individual with lung disease; Franciosi *et al.*, 2019: 1 individual with lung disease; Joly *et al.*, 2015: 1 individual with lung disease | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| 55 | **PI*M malton/ PI*iiyama** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Cases not reported | | | | | |
| 56 | **PI*M malton/ PI*P lowell** | 0 | 0 | 0.0 | N/A | **Unknown risk** |
| | Clinical cases…