Development of Quality Control Materials for Genetic Testing

Nov 30, 2005Geel Belgium

Lisa Kalman, PhDCoordinator, GTQC

CDCLKalman@cdc.gov

GTQC FlowchartGTQC Flowchart

GTQC WebsiteGTQC Websitewww.phppo.cdc.gov/dls/genetics/qcmaterialswww.phppo.cdc.gov/dls/genetics/qcmaterials

Provides information about:Provides information about:Availability and validation status of a variety of QC Availability and validation status of a variety of QC materialsmaterialsQC material needsQC material needsHow to contribute potential QC materialsHow to contribute potential QC materialsProfessional and regulatory guidanceProfessional and regulatory guidanceReferencesReferencesCDCCDC’’s efforts to improve the quality of genetic testings efforts to improve the quality of genetic testing

Solicits input from the genetic testing communitySolicits input from the genetic testing community

QC Material DevelopmentQC Material Development

QC Materials for Huntington Disease (HD) QC Materials for Huntington Disease (HD) TestingTesting

QC material needs for HD allele sizing QC material needs for HD allele sizing were identified by consultation with clinical were identified by consultation with clinical laboratory directorslaboratory directors

Use 15 preUse 15 pre--existing cell lines from existing cell lines from CoriellCoriell. . Cell culture and DNA preparation Cell culture and DNA preparation -- CoriellCoriell

10 labs have begun verification studies 10 labs have begun verification studies (coordinated by Sue Richards)(coordinated by Sue Richards)

Elaine SpectorUniversity of Colorado School Medicine

Fred SchaefferCenter for Genetic Testing at Saint Francis

Beth RohlfsGenzyme Genetics

Kristy Richie/John Jakupciak*

NIST**Sequence analysis

Sue RichardsOregon Health and Sciences University

Kasinathan MuralidharanEmory University

Karla MattesonUniversity of Tennessee Medical Center

Gerald L. FeldmanWayne State University/DMC University

Brett CaseyChildren,s and Women,s Health Centre of BC Vancouver, Canada

Arlene BullerQuest Diagnostics

Elizabeth M. Berry-KravisRush University Medical Center

ContactLaboratoryHD Verification LabsHD Verification Labs

Large allele/Normal86/22

Large allele/Normal69/17

Large allele/Normal63/22

Good for sizing 40-50/Low-end expansion50/37

Two expanded alleles47/44

Low-end expansion/smallest40/15

Low-end expansion39/22

Low-end expansion/normal36/17

Borderline/affected35/4529/1524/15

Closely spaced normal alleles21/20

Closely spaced normal alleles18/17

Homozygous normal15/15

Reason(CAG)n Sized bysubmitter

HD QC Material PanelHD QC Material Panel

Summary of HD Verification ProtocolEach lab will receive 50µg aliquots from the 14 HD lines and from a previously verified line (control)Each laboratory will test the panel and the control using their in-house assayThree separate runs will be performed on each sample, each on a separate dayLabs will send repeat size and raw data to Sue Richards, who will

Examine data qualityCheck for discrepanciesCompile and anonymize data, forward to CDC

Kristy Richie, John Jakupciak (NIST) - sequence analysisCDC - statistical analysis and report results on websiteRepeat sizes - expressed as an estimate with a 95% confidence interval

HD Verification HD Verification –– Current StatusCurrent Status

CoriellCoriell prepared DNA from all 14 cell prepared DNA from all 14 cell lines and shipped to labslines and shipped to labs

Most (all?) labs have received their DNA Most (all?) labs have received their DNA samples and are assaying themsamples and are assaying them

Kristy and John are working on sequence Kristy and John are working on sequence proceduresprocedures

HD HD –– Next StepsNext Steps

Analyze dataAnalyze data

Post data on websitePost data on website

Publication of studyPublication of study

Fragile X workgroup has identified fragile X QC needsFragile X workgroup has identified fragile X QC needs

Cell lines containing fragile X alleles of interest were Cell lines containing fragile X alleles of interest were obtained from collections at obtained from collections at CoriellCoriell Cell Repositories and Cell Repositories and from Dr. Sherman at Emory Universityfrom Dr. Sherman at Emory University

Cell culture and DNA preparationCell culture and DNA preparation-- CoriellCoriellAdditional alleles needed Additional alleles needed

Verification project sponsored by The Association of Verification project sponsored by The Association of Molecular Pathologists (AMP)Molecular Pathologists (AMP)

Verification studies have begun in 9 labs (Fragile XVerification studies have begun in 9 labs (Fragile X--pertsperts+ AMP, Jean Amos+ AMP, Jean Amos--Wilson, Coordinator)Wilson, Coordinator)

QC Materials for Fragile XQC Materials for Fragile X

Premutation120Normal/Premutation30/120Normal/ Full Mutation23/200

Normal/Premutation30/100Premutation100Normal/Premutation30/85Premutation79Top of Gray zone31/55Top of Gray zone54Normal/Gray zone31/46Gray zone46Normal/Gray zone29/45

4129/41

Closely spaced normal29/30Homozygous normal29/29Reason(CGG)n sized by

submitter

Fragile X Fragile X QC PanelQC Panel

Fragile X Verification Labs

Andrea Ferriera-GonzalezVirginia Commonwealth University

Larry SilvermanUniversity of Virginia Health Sciences Center

Sue RichardsOregon Health & Science University

Tom PriorOhio State UniversityKristy Richie/J. JakupciakNIST*Ed HighsmithMayo ClinicBeth RohlfsGenzyme Genetics

Kasinathan MuralidharanEmory University School of Medicine

Ben RoaBaylor College of MedicineElaine LyonARUP Laboratories

ContactLaboratory

Summary of Fragile X Summary of Fragile X Verification ProtocolVerification Protocol

Each lab will receive 50Each lab will receive 50µµg aliquots from the 15 fragile X lines g aliquots from the 15 fragile X lines and from 5 previously verified lines (controls)and from 5 previously verified lines (controls)

Each laboratory will test the panel and the controls using theirEach laboratory will test the panel and the controls using theirinin--house PCRhouse PCR--based assaybased assay

Three separate runs will be performed on each sample, each Three separate runs will be performed on each sample, each on a separate day. on a separate day. SouthernsSoutherns will be done once.will be done once.

Labs will also test the fragile X DNA 3 times using a prototype Labs will also test the fragile X DNA 3 times using a prototype fragile X assay from Celera Diagnosticsfragile X assay from Celera Diagnostics

Labs will send repeat size and raw data to Jean AmosLabs will send repeat size and raw data to Jean Amos--Wilson, Wilson, who willwho will

Examine data qualityExamine data qualityCheck for discrepanciesCheck for discrepanciesCompile and Compile and anonymizeanonymize data, forward to CDCdata, forward to CDC

Summary of Fragile X Summary of Fragile X Verification Protocol, cont.Verification Protocol, cont.

Kristy Richie, John Jakupciak (NIST) Kristy Richie, John Jakupciak (NIST) -- sequence sequence analysis of male linesanalysis of male lines

CDC/AMP CDC/AMP -- statistical analysis and report results on statistical analysis and report results on website and publicationwebsite and publication

Repeat sizes Repeat sizes -- expressed as an estimate with a 95% expressed as an estimate with a 95% confidence intervalconfidence interval

DNA/cell lines available from DNA/cell lines available from CoriellCoriell Cell Cell RepositoriesRepositories

QC materials for Ashkenazi QC materials for Ashkenazi Jewish (AJ) PanelJewish (AJ) Panel

ACOG recommends carrier screening for genetic ACOG recommends carrier screening for genetic diseases in individuals with Ashkenazi Jewish diseases in individuals with Ashkenazi Jewish descent descent -- 20042004

Screening is recommended forScreening is recommended forTayTay--sachssachs diseasediseaseCanavanCanavan diseasediseaseCystic fibrosisCystic fibrosisFamilial Familial dysautonomiadysautonomiaMucolipidosisMucolipidosis IVIVNiemannNiemann--Pick disease type APick disease type AFanconiFanconi anemia group Canemia group CBloom syndrome Bloom syndrome GaucherGaucher diseasedisease

The ACOG recommendations specify disorder, The ACOG recommendations specify disorder, but not mutations to be tested.but not mutations to be tested.

So, whatSo, what’’s on the menu?s on the menu?

Ashkenazi Jewish (AJ) QC Ashkenazi Jewish (AJ) QC Material SelectionMaterial Selection

Compiled list of disorders/mutations tested Compiled list of disorders/mutations tested at a number of clinical labsat a number of clinical labs

Compared to cell lines available at Compared to cell lines available at CoriellCoriell

Chose available cell lines to develop AJ Chose available cell lines to develop AJ QC panelQC panel

QC Materials for AJ PanelQC Materials for AJ Panel31 31 CoriellCoriell Cell lines homozygous or heterozygous for Cell lines homozygous or heterozygous for

disease causing mutations disease causing mutations -- 9 disorders:9 disorders:

TayTay--sachssachs diseasediseaseCanavanCanavan diseasediseaseGlycogen storage diseaseGlycogen storage diseaseFamilial Familial dysautonomiadysautonomiaMucolipidosisMucolipidosis IVIVNiemannNiemann--Pick disease type APick disease type AFanconiFanconi anemia group Canemia group CBloom syndrome Bloom syndrome GaucherGaucher diseasedisease

have been chosen for QC material developmenthave been chosen for QC material development

QC materials for AJ PanelQC materials for AJ Panel

Cell culture and DNA preparation from 31 Cell culture and DNA preparation from 31 cell lines containing many of the needed cell lines containing many of the needed mutations mutations -- CoriellCoriell

Materials with other mutations are needed Materials with other mutations are needed

Verification (sequence analysis and multiVerification (sequence analysis and multi--laboratory evaluation) begins soon! laboratory evaluation) begins soon! (to be (to be coordinated by Kasinathan Muralidharan, Emory coordinated by Kasinathan Muralidharan, Emory UniversityUniversity))

Assessment of Needs for Cystic Assessment of Needs for Cystic Fibrosis (CF) QC MaterialsFibrosis (CF) QC Materials

Assess which mutations are currently Assess which mutations are currently tested in clinical labstested in clinical labs

Compare mutations tested to available Compare mutations tested to available materialsmaterials

Identify mutations for which QC materials Identify mutations for which QC materials need to be developedneed to be developed

CF QC Material NeedsCF QC Material NeedsEvaluation of NeedsEvaluation of Needs

Searched published articles on CF testing Searched published articles on CF testing and ACMG recommendationsand ACMG recommendations

Compiled a list of mutations included in Compiled a list of mutations included in laboratory laboratory –– developed testing and developed testing and commercial test reagentscommercial test reagents

Used Used CoriellCoriell and GTQC (CDC) websites to and GTQC (CDC) websites to identify available genomic materialsidentify available genomic materials

Searched manufacturersSearched manufacturers’’ websites for websites for mutations represented in synthetic control mutations represented in synthetic control materialsmaterials

CF Mutations TestedCF Mutations TestedCollected information about 35 US testing labsCollected information about 35 US testing labs

102 CF mutations tested in US clinical labs102 CF mutations tested in US clinical labs

There are There are CoriellCoriell cell lines for 42 mutationscell lines for 42 mutations60 mutations not represented at 60 mutations not represented at CoriellCoriell

34/35 clinical labs in US include all ACMG34/35 clinical labs in US include all ACMG--23 23 mutationsmutations

Number of cystic fibrosis mutations tested in US labs,

N=35

0

2

4

6

8

10

12

14

16<2

5

25-2

9

30-3

4

35-3

9

40-4

4

45-4

9

50-5

4

55-5

9

60-6

4

65-6

9

70-7

4

75-7

9

80-8

4

85-8

9

Number of mutations tested

Num

ber o

f lab

s

Availability of CF Testing QC Materials

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Genomic Synthetic Both None

Type of control material available

Perc

enta

ge o

f mut

atio

ns

cove

red Not verified

Verified

N = 102

51

3342

28

14

What to consider when identifying CF What to consider when identifying CF QC material priorities?QC material priorities?

Number of labs testing for the Number of labs testing for the mutationmutation??Number of commercial reagents Number of commercial reagents including the mutationincluding the mutation??Frequency of the mutation in the panFrequency of the mutation in the pan--ethnic U.S. populationethnic U.S. population??Mutations with high frequencies in the Mutations with high frequencies in the African American and Hispanic African American and Hispanic populationspopulations??

CF CF –– Current SituationCurrent Situation

CoriellCoriell CF linesCF linesThe ACMG 23 mutation panel is The ACMG 23 mutation panel is represented at represented at CoriellCoriell6 mutations on the panel have been 6 mutations on the panel have been verified, the rest have notverified, the rest have notMany commonly tested mutations are not Many commonly tested mutations are not represented by represented by CoriellCoriell cell linescell lines

NIST may sequence to confirm NIST may sequence to confirm mutations in ACMG 25 (23?) Panelmutations in ACMG 25 (23?) Panel

CF CF –– Next StepsNext Steps

Should we try to develop more verified QC Should we try to develop more verified QC materials for CFmaterials for CF??

Which alleles should we verifyWhich alleles should we verify??

Which alleles should we obtainWhich alleles should we obtain??

How do we decide?How do we decide?

Human Subject Research IssuesProtocols were submitted to three academic Protocols were submitted to three academic institutionsinstitutions

Initial protocol involved two sample types:

Anonymized residual blood sample submission Targeted recollection of patient blood samples with informed consent

In all 3 institutions, the In all 3 institutions, the IRB Did not allow use of residual blood, even if anonymized, without patient consentAllowed targeted recollection with informed consent

GTQC GTQC -- Next StepsNext Steps……..

Finish current verification studiesFinish current verification studies

Continue as an information resourceContinue as an information resource

Identify new targets and directions for QC Identify new targets and directions for QC material developmentmaterial development

Address human subject and regulatory issuesAddress human subject and regulatory issues

Coordinate with European QC material Coordinate with European QC material development efforts?development efforts?