TAG-IT CYSTIC FIBROSIS KIT

DEN050004 · Tm Bioscience Corporation · NUA · May 9, 2005 · Immunology

Device Facts

• Record ID: DEN050004
• Device Name: TAG-IT CYSTIC FIBROSIS KIT
• Applicant: Tm Bioscience Corporation
• Product Code: NUA · Immunology
• Decision Date: May 9, 2005
• Decision: DENG
• Submission Type: Post-NSE
• Regulation: 21 CFR 866.5900
• Device Class: Class 2
• Attributes: Software as a Medical Device, Pediatric

Intended Use

The Tag-It™ Cystic Fibrosis Kit is a device used to simultaneously detect and identify a panel of mutations and variants in the cvstic fibrosis transmembrane conductance regulator (CFTR) gene in human blood specimens. The panel includes mutations and variants currently recommended by the American College of Medical Genetics and American College of Obstetricians and Gynecologists (ACMG/ACOG), plus some of the worlds most common and North American-prevalent mutations. The Tag-It™ Cystic Fibrosis Kit is a qualitative genotyping test which provides information intended to be used for carrier testing in adults of reproductive age, as an aid in newborn screening, and in confirmatory diagnostic testing in newborns and children. The kit is not indicated for use in fetal diagnostic or pre-implantation testing. This kit is also not indicated for stand-alone diagnostic purposes.

Device Story

The Tag-It™ Cystic Fibrosis Kit is an in vitro diagnostic test for detecting 39 mutations and 4 polymorphisms in the CFTR gene from purified human genomic DNA. The assay uses multiplex PCR followed by multiplex allele-specific primer extension (ASPE) with biotinylated dCTP. Extended primers are hybridized to spectrally distinguishable, bead-immobilized anti-tags and detected via flow cytometry using a Luminex® 100 xMAP™ system. The Tag-It™ Data Analysis Software (TDAS CF-I) processes the fluorescent signals to determine genotypes. The device is used in clinical laboratories by trained personnel. Results are interpreted by healthcare providers in the context of clinical status and testing algorithms to aid in carrier screening and diagnostic confirmation. It benefits patients by identifying CFTR mutations associated with cystic fibrosis or infertility, facilitating genetic counseling and risk assessment.

Clinical Evidence

Bench testing only. Analytical performance established using 828 replicates of Coriell genomic samples and synthetic controls. Reproducibility >99.99% across 3 sites, 3 lots, and multiple operators/instruments. Accuracy demonstrated by 100% concordance with bi-directional dideoxy terminal DNA sequencing for 68 clinical samples and additional genomic/synthetic controls. 95% CI for accuracy: 99.9%–100%.

Technological Characteristics

Multiplex PCR and ASPE assay. Components: Multiplex PCR primer mix, ASPE primer mix, 5.0 micron polystyrene beads dyed with red/infrared fluorochromes, 10X wash buffer, and TDAS CF-I software. Detection via Luminex® 100 xMAP™ flow cytometry. Standards referenced: CLSI MM01-A, EP5-A, EP12-A; ACMG/ACOG guidelines. Storage: -25°C to -15°C.

Indications for Use

Indicated for carrier testing in adults of reproductive age, as an aid in newborn screening, and in confirmatory diagnostic testing in newborns and children. Not for fetal diagnostic, pre-implantation, or stand-alone diagnostic use. Prescription use only.

Regulatory Classification

Identification

The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It is intended as an aid in confirmatory diagnostic testing of individuals with suspected cystic fibrosis (CF), carrier identification, and newborn screening. This device is not intended for stand-alone diagnostic purposes, prenatal diagnostic, pre-implantation, or population screening.

Special Controls

*Classification.* Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: CFTR Gene Mutation Detection System.” See § 866.1(e) for the availability of this guidance document.

Submission Summary (Full Text)

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#### 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

### A. 510(k) Number:

k043011

### B. Purpose for Submission:

New device

### C. Measurand:

CFTR (cystic fibrosis transmembrane conductance regulator) gene from human blood specimens

### D. Type of Test:

Multiplex PCR followed by multiplex allele specific primer extension for genotyping, hybridized to multiplexed fluorescing microparticles, detected by flow cytometry

### E. Applicant:

Tm Bioscience Corporation

# F. Proprietary and Established Names:

Tag-ItTM Cystic Fibrosis Kit

#### G. Regulatory Information:

- 1. Regulation section:
21 CFR 866.5900, CFTR (cystic fibrosis transmembrane conductance regulator) gene mutation detection system

- 2. Classification: Class II
- 3. Product code:

NUA, System, test, CFTR (cystic fibrosis transmembrane conductance regulator) gene mutation detection

- 4. Panel: Immunology (82)
# H. Intended Use:

- 1. Intended use(s):
The Tag-It™ Cystic Fibrosis Kit is a device used to simultaneously detect and identify a panel of mutations and variants in the cvstic fibrosis transmembrane conductance regulator (CFTR) gene in human blood specimens. The panel includes mutations and variants currently recommended by the American College of Medical Genetics and American College of Obstetricians and Gynecologists (ACMG/ACOG), plus some of the worlds most common and North American-prevalent mutations. The Tag-It™ Cystic Fibrosis Kit is a qualitative genotyping test which provides information intended to be used for carrier testing in adults of reproductive age, as an aid in newborn screening, and

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in confirmatory diagnostic testing in newborns and children.

The kit is not indicated for use in fetal diagnostic or pre-implantation testing. This kit is also not indicated for stand-alone diagnostic purposes.

- 2. Indication(s) for use: Same as intended use.
- 3. Special conditions for use statement(s): For prescription use only.

Since the Tag-It™ Cystic Fibrosis Kit detects a limited number of mutations (out of more than 1300 mutations identified in the CFTR gene), it should not be used alone to diagnose cystic fibrosis. Assay results should be interpreted only in the context of the overall testing algorithm and clinical status of the patient.

Carrier screening for CF using molecular testing has been recommended by the NIH and the ACMG/ACOG for reproductive couples of all ethnicities, individuals with a family history of CF, and pregnant women. When used in CF carrier detection, the Tag-It™ Cystic Fibrosis Kit may identify: (i) positive/positive, positive/negative, and negative/negative couples: (ii) individuals who have a family history of CF; (iii) otherwise healthy males who carry mutations or variants associated with infertility. Interpretation of results will depend on the patient demographics, and must take into consideration that some mutations are more common in certain populations.

Clinical variability of the disease based on the genotype-phenotype correlation variation for different mutations may result in the need for genetic counseling. Genetic counseling will enable individuals and couples to receive accurate information about risks and prognostic factors.

#### 4. Special instrument requirements:

Luminex 100 IS (Integrated System); other names used: Luminex® 100 xMAPTM System.

#### I. Device Description:

The Tag-It™ Cystic Fibrosis Kit includes the following components:

- Multiplex PCR Primer Mix including dNTPs designed to simultaneously produce 16 . amplimers of the CFTR gene
- . Multiplex ASPE Primer Mix including dNTPs (86 primers designed to hybridize to either wild-type or mutant alleles with proprietary sequences at their 5' ends designed to specifically hybridize to complementary sequences coupled to the bead component of the kit)
- Coupled Bead Suspension (86 spectrally distinguishable populations of 5.0 micron . polystyrene beads internally dyed with red and infrared fluorochromes coupled to proprietary DNA sequences designed to specifically hybridize to complementary sequences on the ASPE primers)

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- 10X Wash Buffer
- . Tag-It™ Data Analysis Software (TDAS CF-I)

### J. Substantial Equivalence Information:

- 1. Predicate device name(s): None
- 2. Predicate 510(k) number(s): None
- 3. Comparison with predicate: Not applicable

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

- American College of Medical Genetics (ACMG)/American College of Obstetricians and Gynecologists
- 2001, 2002, 2004 ACMG Technical Standards and Guidelines for CFTR Mutation o Testing
- ACMG 2004 Standards and Guidelines for Clinical Genetics Laboratories o
- 2004 Cystic Fibrosis Foundation (CFF) / Center for Disease Control (CDC) ● Recommendations on Newborn Screening for Cystic Fibrosis
- CLSI Guidances
- o MM01-A: Molecular Diagnostic Methods for Genetic Diseases
- EP5-A: Evaluation of Precision Performance of Clinical Chemistry Devices; o Approved Guideline.
- o EP12-A: User Protocol for Evaluation of Qualitative Test Performance: Approved Guideline.
- FDA Guidances ●
- CDRH Draft Guidance for Industry and FDA Reviewers titled: Multiplex Tests o for Heritable DNA Markers, Mutations and Expression Patterns, Feb 27, 2003.
- CDRH Draft Guidance for Industry and FDA Reviewers titled: Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests, Mar 12. 2003.
- 0 CDRH Guidance for Industry and FDA Reviewers titled: Guidance for the content of premarket submissions for software contained in medical devices, May 29. 1998.
- 0 CDRH Guidance for Industry and FDA Staff titled: General Principles of Software Validation, Jan 11, 2002.

# L. Test Principle:

The Tag-It™ Cystic Fibrosis Kit tests for 39 mutations and 4 polymorphisms in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. These mutations include those currently recommended for testing by the ACMG/ACOG (denoted with an asterisk in the list below) plus 16 mutations shown to be associated with CF phenotypes in Caucasian Americans, Hispanic Americans and African Americans:

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| ΔF508* | 1717-1G>A* | W1282X* | 2307insA |
|-----------|---------------|-----------|----------|
| ΔI507* | R560T* | N1303K* | Y1092X |
| G542X* | R553X* | 394delTT | M1101K |
| G85E* | G551D* | Y122X | S1255X |
| R117H* | 1898+1G>A* | R347H | 3876delA |
| 621+1G>T* | 2184delA* | V520F | 3905insT |
| 711+1G>T* | 2789+5G>A* | A559T | 5/7/9T |
| 1078delT | 3120+1G>A* | S549N | F508C |
| R334W* | R1162X* | S549R | I507V |
| R347P* | 3659delC* | 1898+5G>T | I506V |
| A455E* | 3849+10kbC>T* | 2183AA>G | |

After sample preparation of genomic DNA (which is not a part of the Tag-It™ assay), a multiplex PCR reaction is carried out under optimized conditions. Multiplex allele-specific primer extension (ASPE) is then used for genotyping. In this step, each allele (wild-type or mutant) is detected by a primer with a unique DNA sequence (tag) at its 5' end. Each mutant locus has two allele-specific primers (ASPs), or three for a tri-allelic locus. For each ASP, the 3' end of the primer is a perfect match for its allele, but will have a 3' mismatch on any other allele. A DNA polymerase is used that will only extend the primer when there is a perfect match on the 3' end, so that the primer is only extended if its target allele is present in the sample. Biotin-dCTP is incorporated into the extending chain if extension occurs. After ASPE, the reaction is added directly to microwells containing bead-immobilized oligonucleotides (anti-tags) which are the complements of the DNA tags on the allele specific primers. The beads which contain the anti-tags are spectrally distinguishable from each other. A fluorescent reporter molecule (streptavidin-phycoerythrin) is bound to the biotin on the extended primers. Each tagged primer hybridizes only to its unique anti-tag complement; therefore, each colored bead represents a specific allele, through the bead/antitag/tagged primer association. The beads are then analyzed by the Luminex® xMAP machine. The xMAP" instrument contains two lasers: one identifies the color-coded bead, and the other identifies the presence or absence of extended allele specific primer through the phycoerythrin reporter. Thus, the genotype of that locus is identified by the presence of phycoerythrin signal attached to one or both ASPs.

All mutations and polymorphisms are genotyped in a single multiplex reaction. The data generated by the xMAP" instrument is analyzed by the Tag-It™ Data Analysis Software (TDAS CF-I) to provide a final genotype for the sample.

#### M. Performance Characteristics (if/when applicable):

- 1. Analytical performance:
- a. Precision/Reproducibility:

The Tag-It™ Cystic Fibrosis Kit has a reproducibility of >99.99%. This performance characteristic was established by testing 3 lots of the kit at each of 3 sites (1 internal, 2 external sites, at least 2 operators at each site), over multiple days, using 4 Luminex® systems. The samples tested were genomic samples obtained from the Coriell Cell Repository (Camden, NJ) which were sequenced at all loci probed by the Tag-It™ assay. For each Tag-It™ run, samples were assayed in quadruplicate. Table

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1 below summarizes the genotypes of the samples tested, the number of replicates tested, the calls confirmed by sequencing and the missed calls. Of the 828 replicates tested, there were 7 replicates requiring re-runs due to a sample failure resulting from inadequate signal. TDAS CF-I generates a Sample Failure if one of the probed loci has signals below a predefined threshold. In this situation, TDAS CF-I does not provide genotyping calls for any of the mutations/variants probed in that sample. After these 7 replicates were re-run, all calls were made correctly,

Each of the 828 replicates tested were assayed at 43 loci by the Tag-It™ Cystic Fibrosis Kit, generating a total of 35,604 calls. Of these, 35,602 (i.e. reproducibility >99.99%) were confirmed by sequencing. The 2 missed calls are highlighted in the table below. In one case, the expected call for the R560T locus was HET but TDAS CF-I generated a NO CALL the first time that one of the 36 replicates of that sample was run. TDAS CF-I generates a NO CALL when the allelic ratio does not fall within pre-defined ranges for either a WT, HET, or Mu D call. When this replicate was re-run, the expected call for this locus (i.e. R560T HET) was made, consistent with the calls made for all other replicates of that sample. In the second case, the expected call for AF508 was HET whereas TDAS generated a Mu D call the first time that one of the 36 replicates of that sample was run. When this replicate was re-run, the expected call for (i.e. ΔF508 HET) was made, consistent with the calls made for all other replicates of that sample.

| Purified Genomic Samples:<br>Mutations and Variants<br>detected by bi directional,<br>dideoxy terminal DNA<br>sequencing (all other probed<br>loci were WT) | | Number of Sample<br>Replicates Assayed<br>by the Tag-It™<br>Cystic Fibrosis Kit<br>(3 kit lots used at<br>each of the 3 sites) | | Number of Confirmed Tag-It ™™<br>Calls out of a total of 516 per<br>sample (43 calls per sample x 4<br>replicates x 3 lots) per site | | | |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------|--------------------------------------------------------------------------------------------------------------------------------|-----------|--------------------------------------------------------------------------------------------------------------------------------------|-----------|-----------|-----------------|
| Sample Genotype | Site<br>A | Site<br>B | Site<br>C | Site<br>A | Site<br>B | Site<br>C | Missed<br>Calls |
| ΔF508/ΔF508/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| R553X/ΔF508/7T/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| 3659delC/ΔF508/7T/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| R560T/ΔF508/7T/9T | 12 | 12 | 12 | 515 | 516 | 516 | 1a |
| N1303K/G1349D /7T/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| R334W/7T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| 3120+1G>A/621+1G>T /7T/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| G542X/7T/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| G542X/G542X /9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| M1101K/M1101K /7T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| 711+1G->T(T)/621+1G->T /7T/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| G551D/7T/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |

| Table 1: Reproducibility of the Tag-It™ Cystic Fibrosis Kit (>99.99%: site-to-site, lot-to-lot, | | | | |
|-------------------------------------------------------------------------------------------------|--|--|--|--|
| operator to operator) | | | | |

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| Purified Genomic Samples:<br>Mutations and Variants<br>detected by bi directional,<br>dideoxy terminal DNA<br>sequencing (all other probed<br>loci were WT) | Number of Sample<br>Replicates Assayed<br>by the Tag-It™<br>Cystic Fibrosis Kit<br>(3 kit lots used at<br>each of the 3 sites) | Number of Confirmed Tag-It ™<br>Calls out of a total of 516 per<br>sample (43 calls per sample x 4<br>replicates x 3 lots) per site | | | | | |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------|----|-----|-----|-----|----|
| W1282X/5T/7T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| ΔI507/7T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| 621+1G->T/ΔF508/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| G85E/621+1G->T /7T/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| A455E/ΔF508/9T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| 2789+5G->A/2789+5G->A /7T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| 3849+10C->T/3849+10C->T /7T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| 1717-1G->T(A)/7T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| R1162X/7T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| R347P/G551D /7T | 12 | 12 | 12 | 516 | 516 | 516 | 0 |
| R117H/ΔF508/5T/9T | 12 | 12 | 12 | 516 | 516 | 515 | 1° |

One replicate at Site A had a No Call for R560T. The expected call (R560T HET) was made after re-run. a)

One replicate at Site C had a Mu D call for ΔF508. The expected call (ΔF508 HET) was made after re-run. b)

> The Tag-It™ Cystic Fibrosis Kit detects all mutant/variant/wild-type alleles of the 43 loci assayed with a precision of >99.99%. This performance characteristic has been defined based on an analysis of all genotyping calls (WT, HET, Mu D, variant detected) that can be made at each locus probed by the Tag-It™ Cystic Fibrosis Kit. Table 2 below summarizes data from a study designed according to the CLSI EP5-A guideline. In that study, variation in allelic ratio (AR) values was assessed from results derived from day-to-day (a total of 5 days), user-to-user (a total of 5 users), lot-to-lot (a total of 3 lots of Tag-It™ reagents), reagent-to-reagent (a total of 3 lots of each of the 4 ancillary reagents used to perform the assay), and machine-to-machine (including 3 thermocyclers and 3 Luminex® systems) analyses. AR variation was evaluated both within and between Tag-It™ runs using replicates of a Coriell genomic sample and sequenced synthetic controls (see below) representing all genotyping calls that can be made for all 43 loci that are included on the Tag-It™ panel. Depending on the experiment, between 60 and 240 calls (WT, HET, Mu D. and variant detected) were compared per locus. The repeatability of calls is shown in Table 6 below. Overall, results of this study show that all genotyping calls that can be made by the Tag-It™ Cystic Fibrosis Kit can be made correctly and reproducibly across days, users, reagent lots (Tag-It™ and ancillary) and machines (Luminex® systems and thermocyclers).

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Table 2: Precision of the Tag-It™ Cystic Fibrosis Kit (>99.9% repeatability between days, users, reagents, machines)

| | % correct calls made (WT, HET, Mu D, variant detected) for each locus on the Tag-ItTM panela | | | | | |
|-----------------------|----------------------------------------------------------------------------------------------|-------------------------------|------------------------------------------------|-----------------------------------------------------------|--------------------------------------------------|----------------------------------------|
| 43 loci tested | Day<br>to<br>Day<br>5 days | User<br>to<br>User<br>5 users | Lot to Lot<br>(Tag-ItTM<br>reagents)<br>3 lots | Reagent to<br>Reagent<br>(3 ancillary)c<br>3 lots of each | Thermocycler<br>to<br>Thermocycler<br>3 machines | Luminex<br>to<br>Luminex<br>3 machines |
| G85E | 100% | 99.6%b | 100% | 100% | 100% | 100% |
| 394delTT | 100% | 100% | 100% | 100% | 100% | 100% |
| R117H | 100% | 100% | 100% | 100% | 100% | 100% |
| Y122X | 100% | 100% | 100% | 100% | 100% | 100% |
| 621+1G>T | 100% | 100% | 100% | 100% | 100% | 100% |
| 711+1G>T | 100% | 100% | 100% | 100% | 100% | 100% |
| 1078delT | 100% | 100% | 100% | 100% | 100% | 100% |
| R334W | 100% | 100% | 100% | 100% | 100% | 100% |
| R347P/R347H | 100% | 100% | 100% | 100% | 100% | 100% |
| A455E | 100% | 100% | 100% | 100% | 100% | 100% |
| dl507/ dF508 | 100% | 100% | 100% | 100% | 100% | 100% |
| V520F | 100% | 100% | 100% | 100% | 100% | 100% |
| 1717-1G>A | 100% | 100% | 100% | 100% | 100% | 100% |
| G542X | 100% | 100% | 100% | 100% | 100% | 100% |
| S549N | 100% | 100% | 100% | 100% | 100% | 100% |
| S549R (T>G) | 100% | 100% | 100% | 100% | 100% | 100% |
| G551D | 100% | 100% | 100% | 100% | 100% | 100% |
| R553X | 100% | 100% | 100% | 100% | 100% | 100% |
| A559T | 100% | 100% | 100% | 100% | 100% | 100% |
| R560T | 100% | 100% | 100% | 100% | 100% | 100% |
| 1898+1G>A | 100% | 100% | 100% | 100% | 100% | 100% |
| 1898+5G>T | 100% | 100% | 100% | 100% | 100% | 100% |
| 2183AA>G | 100% | 100% | 100% | 100% | 100% | 100% |
| 2184delA | 100% | 100% | 100% | 100% | 100% | 100% |
| 2307insA | 100% | 100% | 100% | 100% | 100% | 100% |
| 2789+5G>A | 100% | 100% | 100% | 100% | 100% | 100% |
| 3120+1G>A | 100% | 100% | 100% | 100% | 100% | 100% |
| Y1092X | 100% | 100% | 100% | 100% | 100% | 100% |
| M1101K | 100% | 99.6%b | 100% | 100% | 100% | 100% |
| R1162X | 100% | 100% | 100% | 100% | 100% | 100% |
| 3659delC | 100% | 100% | 100% | 100% | 100% | 100% |
| S1255X | 100% | 100% | 100% | 100% | 100% | 100% |
| 3849+10kbC>T | 100% | 100% | 100% | 100% | 100% | 100% |
| 3876delA | 100% | 100% | 100% | 100% | 100% | 100% |
| 3905insT | 100% | 100% | 100% | 100% | 100% | 100% |
| W1282X | 100% | 100% | 100% | 100% | 100% | 100% |
| N1303K | 100% | 100% | 100% | 100% | 100% | 100% |
| 5T/7T/9T | 100% | 100% | 100% | 100% | 100% | 100% |
| I506V/I507V/<br>F508C | 100% | 100% | 100% | 100% | 100% | 100% |

Depending on the experiment, between 60 and 240 calls (WT, HET, Mu D, and variant detected) were a) compared per locus using sequenced genomic and synthetic samples

b) On one occasion (out of 240) for each of M1101K and G85E, there was a "No Call" resulting from low signal which caused ARs to fall outside of the pre-defined ranges for making either a WT, HET or Mu D genotyping call.

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- 3 separate experiments, each with the same lot of the Tag-It™ Cystic Fibrosis Kit but with 3 different lots of c) Taq, Tsp, and SAP, respectively.
- b. Linearity/assay reportable range: Not applicable.
- c. Traceability, Stability, Expected values (controls, calibrators, or methods): Stability studies on the Tag-It™ Cystic Fibrosis Kit support a shelf-life of 1 year when kit reagents are stored at -25℃ to -15℃. Inadvertent temporary exposure (up to 6 days) at temperatures exceeding the recommended storage temperature (up to 37°C) and repeated freeze-thaw cycles (up to 3) will not compromise the integrity of the Tag-It™ Cystic Fibrosis Kit.
- d. Detection limit:

Although the recommendations of the insert are that 25 ng of extracted DNA be used for the PCR reaction described above. it has been established with purified genomic samples that, when the Tag-It™ assay is run as per the methods described, samples with input values ranging between 1 ng and 200 ng provide the correct genotyping calls. It is recommended that the DNA sample be extracted from whole blood (EDTA or citrate) using methodologies which provide purified genomic DNA with an ultraviolet light absorbance ratio at 260/280 greater than 1.7.

- e. Analytical specificity:
#### Interfering Substances

The Tag-It™ Cystic Fibrosis Kit can be used to detect mutations in the CFTR gene using genomic DNA isolated from blood with a sufficient purity. i.e., with the ratio of absorbance at 260 nm vs. that at 280 nm being 1.7-2.0. Interference studies were not conducted since input into the assay is purified genomic DNA and there is sufficient data in the literature to support a recommendation that the DNA sample be extracted from whole blood (EDTA or citrate) using methodologies which provide genomic DNA with an ultraviolet light absorbance ratio at 260/280 which is greater than 1.7.

- f. Assay cut-off: Not applicable.
#### 2. Comparison studies:

- a. Method comparison with predicate device:
- All genotyping calls made by the Tag-It™ Cystic Fibrosis Kit have been compared to those made by bi-directional dideoxy terminal DNA sequencing in clinical samples or, in cases of rare alleles for which clinical samples were not available, in replicates of purified genomic samples obtained from the Coriell Institute (Camden, NJ) and synthetic oligonucleotide controls. Concordance of calls for the 39 disease causing mutations and 4 variants included on the Tag-It™ panel are provided in Table 3 below as percent agreements. All results summarized in Table 3 below are from studies in which the input DNA was equivalent to the concentration recommended in this insert (25 ng). Synthetic controls were prepared by adding the DNA containing the mutation at a copy number equivalent to a natural sample blended in a genomic

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DNA matrix to represent all possible calls (WT, HET, Mu D, variant detected) for each mutation and variant probed for by the Tag-It™ Cystic Fibrosis Mutation Detection Kit. Not included in the table below are clinical samples for which DNA sequencing was unable to provide an unambiguous genotyping call for the particular locus in question.

| Mutations /<br>Variants | Number of Samples in which the wild-type allele was identified | | | | Number of Samples in which the mutant/variant allele was identified | | | | Percent<br>Agreement<br>All Samples |
|-------------------------|--------------------------------------------------------------------|----------|-----------------------|-----------------------|------------------------------------------------------------------------|----------|-----------------------|-----------------------|-------------------------------------|
| | Total | Clinical | Genomic<br>replicates | Synthetic<br>controls | Total | Clinical | Genomic<br>replicates | Synthetic<br>controls | |
| G85E | 695 | 137 | 540 | 18 | 83 | 1 | 36 | 46 | 100% |
| 394delTT | 973 | 139 | 828 | 6 | 48 | 0 | 0 | 48 | 100% |
| R117H | 953 | 125 | 792 | 36 | 77 | 13 | 36 | 28 | 100% |
| Y122X | 973 | 139 | 828 | 6 | 30 | 0 | 0 | 30 | 100% |
| 621+1G>T | 834 | 132 | 684 | 18 | 195 | 7 | 144 | 44 | 100% |
| 711+1G>T | 940 | 130 | 792 | 18 | 84 | 2 | 36 | 46 | 100% |
| 1078delT | 936 | 102 | 828 | 6 | 59 | 1 | 0 | 58 | 100% |
| R334W | 926 | 98 | 792 | 36 | 69 | 5 | 36 | 28 | 100% |
| R347P | 898 | 100 | 792 | 6 | 75 | 3 | 36 | 36 | 100% |
| R347H | 937 | 103 | 828 | 6 | 40 | 0 | 0 | 40 | 100% |
| A455E | 953 | 137 | 792 | 24 | 77 | 1 | 36 | 40 | 100% |
| ΔI507 | 953 | 137 | 792 | 24 | 50 | 2 | 36 | 12 | 100% |
| ΔF508 | 666 | 66 | 576 | 24 | 337 | 73 | 252 | 12 | 100% |
| V520F | 1009 | 139 | 828 | 42 | 24 | 0 | 0 | 24 | 100% |
| 1717-1G>A | 969 | 129 | 792 | 48 | 63 | 9 | 36 | 18 | 100% |
| G542X | 932 | 128 | 756 | 48 | 100 | 10 | 72 | 18 | 100% |
| S549N | 972 | 138 | 828 | 6 | 16 | 0 | 0 | 16 | 100% |
| S549R (T>G) | 990 | 138 | 828 | 24 | 12 | 0 | 0 | 12 | 100% |
| G551D | 955 | 127 | 792 | 36 | 95 | 11 | 72 | 12 | 100% |
| R553X | 956 | 134 | 792 | 30 | 58 | 4 | 36 | 18 | 100% |
| A559T | 997 | 139 | 828 | 30 | 18 | 0 | 0 | 18 | 100% |
| R560T | 972 | 132 | 792 | 48 | 59 | 5 | 36 | 18 | 100% |
| 1898+1G>A | 969 | 135 | 828 | 6 | 32 | 4 | 0 | 28 | 100% |
| 1898+5G>T | 1003 | 139 | 828 | 36 | 30 | 0 | 0 | 30 | 100% |
| 2183AA>G | 973 | 139 | 828 | 6 | 34 | 0 | 0 | 34 | 100% |
| 2184delA | 973 | 139 | 828 | 6 | 36 | 0 | 0 | 36 | 100% |
| 2307insA | 1003 | 139 | 828 | 36 | 28 | 0 | 0 | 28 | 100% |
| 2789+5G>A | 946 | 136 | 792 | 18 | 85 | 3 | 36 | 46 | 100% |
| 3120+1G>A | 939 | 135 | 792 | 12 | 89 | 1 | 36 | 52 | 100% |
| Y1092X | 961 | 127 | 828 | 6 | 58 | 0 | 0 | 58 | 100% |
| M1101K | 947 | 119 | 792 | 36 | 66 | 0 | 36 | 30 | 100% |
| R1162X | 937 | 121 | 792 | 24 | 80 | 4 | 36 | 40 | 100% |
| 3659delC | 942 | 126 | 792 | 24 | 83 | 5 | 36 | 42 | 100% |
| S1255X | 973 | 139 | 828 | 6 | 28 | 0 | 0 | 28 | 100% |
| 3849+10kbC>T | 898 | 94 | 792 | 12 | 96 | 8 | 36 | 52 | 100% |
| 3876delA | 1003 | 139 | 828 | 36 | 30 | 0 | 0 | 30 | 100% |
| 3905insT | 968 | 134 | 828 | 6 | 31 | 1 | 0 | 30 | 100% |
| W1282X | 939 | 111 | 792 | 36 | 74 | 12 | 36 | 26 | 100% |
| N1303K | 901 | 97 | 792 | 12 | 95 | 5 | 36 | 54 | 100% |
| 5T/7T/9T | N/A | N/A | N/A | N/A | 1023 | 137 | 828 | 58 | 100% |
| Mutations /<br>Variants | Number of Samples in which the wild-<br>type allele was identified | | | | Number of Samples in which the<br>mutant/variant allele was identified | | | | Percent<br>Agreement |
| | Total | Clinical | Genomic<br>replicates | Synthetic<br>controls | Total | Clinical | Genomic<br>replicates | Synthetic<br>controls | All Samples |
| | | | | | | | | | |
| I506V | 139 | 139 | 0 | 0 | 12 | 0 | 0 | 12 | 100% |
| I507V | 139 | 139 | 0 | 0 | 12 | 0 | 0 | 12 | 100% |
| F508C | 136 | 136 | 0 | 0 | 14 | 2 | 0 | 12 | 100% |

Table 3: Samples (clinical, genomic replicates and synthetic controls) in which Tag-ft™ calls were compared to sequencing calls

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One replicate of a genomic sample gave a "No Call" for this locus. Upon re-run this replicate gave a HET call, a. consistent with sequencing results.

2 clinical samples were misidentified during transfer to sequencing plates (one sample identified as a WT by the b. Tag-It™ assay at this locus was initially identified as a HET by sequencing and a second sample identified as a HET by the Tag-It™ assay was initially identified as a WT by sequencing, sequencing, sequencing calls at this locus were concordant with Tag-It™ results.

> DNA sequencing provided unambiguous, comparable, genotyping calls at all 43 loci for 68 clinical samples. The mutations and variants detected in these 68 clinical samples are listed in Table 4 below. There were an additional 69 clinical samples genotyped both by the Tag-It™ Cystic Fibrosis Kit and DNA sequencing that were excluded from Table 4 below because DNA sequencing only provided unambiguous calls at a subset of the 43 loci probed. Some possible explanations for ambiguous sequencing results include: 1) poor quality DNA (e.g. impure samples or low concentrations) resulting in a decreased yield in PCR product; 2) PCR products with GC rich sequences; or 3) PCR products forming secondary structures. In those 69 excluded samples, there was 100% concordance with sequencing at the subset of loci which were comparable (Table 3). For the 68 samples included in Table 4, the Tag-It™ Cystic Fibrosis Kit accurately identified all 43 alleles probed, based on 100% concordance with DNA sequencing for all calls made.

Table 4: Accuracy of the Tag-It™ Cystic Fibrosis Kit (100% based on concordance with unambiguous bidirectional dideoxy DNA sequencing at all 43 loci probed, 95% CI: 99.9% - 100%)

| Clinical<br>Sample | Disease causing mutations detected<br>and genotyping call | T-Tract<br>variants | Other<br>variants | Concordant<br>Calls |
|---------------------------------------------------------------|-----------------------------------------------------------|---------------------|-------------------|---------------------|
| 1 | None | detected<br>7T | detected<br>None | 43/43 |
| | | | | |
| 2 | 3659delC HET | 7T/9T | None | 43/43 |
| 3 | R560T HET | 7T | None | 43/43 |
| 4 | R560T HET | 5T/7T | None | 43/43 |
| 5 | G542X HET | 7T/9T | None | 43/43 |
| 6 | VE208 HET | 7T/9T | None | 43/43 |
| 7 | 3849+10kbC>T HET | 7T | F508C | 43/43 |
| 8 | 3849+10kbC>T HET | 71 | None | 43/43 |
| 9 | W1282X HET | 7T | None | 43/43 |
| 10 | W1282X HET | 7T | None | 43/43 |
| 11 | 3849+10kbC>T HET | 7T/9T | F508C | 43/43 |
| 12 | R334W HET | 71 | None | 43/43 |
| 13 | 1891+1G>A HET | 7T | None | 43/43 |
| 14 | R1162X HET | 7T | None | 43/43 |
| 15 | R334W HET | 7T | None | 43/43 |
| 16 | G542X HET | 9T | None | 43/43 |
| 17 | G542X HET | 7T/9T | None | 43/43 |
| 18 | 2789+5G>A HET | 7T | None | 43/43 |
| Clinical | Disease causing mutations detected | T-Tract | Other | Concordant |
| Sample | and genotyping call | variants | variants | Calls |
| | | detected | detected | |
| 19 | 621+1G>T HET | 7T/9T | None | 43/43 |
| 20 | 621+1G>T HET | 7T/9T | None | 43/43 |
| 21 | 1717-1G>A HET | 7T | None | 43/43 |
| 22 | ΔI507 HET | 7T | None | 43/43 |
| 23 | ΔF508 HET | 7T/9T | None | 43/43 |
| 24 | N1303K HET | 7T/9T | None | 43/43 |
| 25 | 1717-1G>A HET | 7T | None | 43/43 |
| 26 | N1303K HET | 7T/9T | None | 43/43 |
| 27 | N1303K HET | 7T/9T | None | 43/43 |
| 28 | G551D HET | 5T/7T | None | 43/43 |
| 29 | G551D HET | 7T | None | 43/43 |
| 30 | G551D HET | 7T | None | 43/43 |
| 31 | 3659delC HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 32 | 3649+10kbC>T and ΔF508 HET | 9T | None | 43/43 |
| 33 | R117H HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 34 | 3659delC HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 35 | R1162X HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 36 | R334W MUT | 7T | None | 43/43 |
| 37 | 711+1G>T HET and 3905insT HET | 7T | None | 43/43 |
| 38 | G542X HET and AF508 HET | 9T | None | 43/43 |
| 39 | 1717-1G>A HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 40 | 347P HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 41 | R117H HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 42 | R117H HET and ΔF508 HET | 5T/9T | None | 43/43 |
| 43 | R117H HET and 1898+1G>A HET | 5T/7T | None | 43/43 |
| 44 | R347P HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 45 | R117H HET and ΔF508 HET | 5T/9T | None | 43/43 |
| 46 | R553X HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 47 | AF508 MUT | 9T | None | 43/43 |
| 48 | AF508 MUT | 9T | None | 43/43 |
| 49 | AF508 MUT | 9T | None | 43/43 |
| 50 | 621+1G>T HET and 3120+1G>A HET | 7T/9T | None | 43/43 |
| 51 | ΔF508 HET and W1282X HET | 7T/9T | None | 43/43 |
| 52 | AF508 MUT | 9T | None | 43/43 |
| 53 | AF508 MUT | 9T | None | 43/43 |
| 54 | R553X HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 55 | ΔF508 HET and R347P HET | 7T/9T | None | 43/43 |
| 56 | N1303K HET and ΔF508 HET | 9T | None | 43/43 |
| 57 | ΔF508 HET and R117H HET | 5T/9T | None | 43/43 |
| 58 | 1717-1G>A HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 59 | 553X HET and AF508 HET | 7T/9T | None | 43/43 |
| 60 | G551D HET and R117H HET | 7T | None | 43/43 |
| 61 | R117H MUT | 5T | None | 43/43 |
| 62 | G542X HET and AF508 HET | 9T | None | 43/43 |
| 63 | G551D HET and AF508 HET | 7T/9T | None | 43/43 |
| 64 | 621+1G>T HET and ΔF508 HET | 9T | None | 43/43 |
| 65 | G542X HET and R117H HET | 5T/9T | None | 43/43 |
| 66 | G551D HET and AF508 HET | 7T/9T | None | 43/43 |
| 67 | 2789+5G>A HET and ΔF508 HET | 7T/9T | None | 43/43 |
| 68 | 1717-1G>A HET and ΔF508 HET | 7T/9T | None | 43/43 |
| Total number of calls concordant with sequencing<br>2924/2924 | | | | |

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

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

| Clinical<br>Sample | Disease causing mutations detected<br>and genotyping call | T-Tract<br>variants<br>detected | Other<br>variants<br>detected | Concordant<br>Calls |
|----------------------------------------|-----------------------------------------------------------|---------------------------------|-------------------------------|---------------------|
| Accuracy: 100% (95% CI: 99.9% to 100%) | | | | |

In summary, Table 5 below demonstrates that there was 100% agreement between the Tag-If™ assay and DNA sequencing on the overall genotype of clinical samples with either 2 identifiable mutations (compound heterozygous samples or homozygous mutant samples), 1 identifiable mutation (heterozygous samples) or no identifiable mutations (wild-type samples). This assessment is based on comparison of genotyping calls at each of the 39 disease causing mutations and 4 variants included on the Tag-It™ panel.

#### Table 5: Agreement on Overall Genotypes of Clinical Samples of calls at 39 loci associated with disease causing mutations

| Tag-It™ Cystic Fibrosis Kit | DNA Sequencing | | Total |
|-----------------------------------------------------|----------------------------------------------------------------|---------------------------------------------------------------------|-------|
| | Clinical Samples with 2 identifiable disease causing mutations | Clinical Samples with 0 or 1 identifiable disease causing mutations | |
| Clinical Samples with 2 identifiable mutations | 38 | 0 | 38 |
| Clinical Samples with 0 or 1 identifiable mutations | 0 | 30 | 30 |
| Total | 38 | 30 | 68 |

b. Matrix comparison: Not applicable

#### 3. Clinical studies:

- Clinical Sensitivity: a.
The clinical…