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CDC Update - GeT-RM ProgramSecond EuroGentest Symposium on

Reference Materials for Genetic TestingMay 15, 2007

Dublin, Ireland

Lisa Kalman, PhDCoordinator, GeT-RM

CDCLKalman@cdc.gov

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Name Change Name Change GeTGeT--RMRM

RM

ReferenceCoordination

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Name ChangeName Change-- GeTGeT--RM?RM?

WHY?WHY?We needed to change the name:We needed to change the name:

To conform to FDA requirementsTo conform to FDA requirements

To harmonize with the efforts of other To harmonize with the efforts of other countriescountries

To reflect that our work is more general than To reflect that our work is more general than QC materials aloneQC materials alone

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Roles of the Roles of the GeTGeT--RM ProgramRM Program

Improve and coordinate information exchange Improve and coordinate information exchange about materials for test development, validation, about materials for test development, validation, control, proficiency testing and researchcontrol, proficiency testing and research

Monitor material needs of the genetic testing Monitor material needs of the genetic testing communitycommunity

Facilitate submission of new cell lines and Facilitate submission of new cell lines and coordinate mutation confirmation studies coordinate mutation confirmation studies

Develop a sustainable community process for Develop a sustainable community process for continued material developmentcontinued material development

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Mutation Confirmation Mutation Confirmation StudiesStudies

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Completed RM Characterization ProjectsCompleted RM Characterization Projects::

Huntington diseaseHuntington disease –– 14 DNA samples14 DNA samplesFragile X Fragile X –– 21 DNA samples21 DNA samplesAshkenazi Jewish Ashkenazi Jewish –– 27 DNA samples 27 DNA samples (21 alleles, 9 disorders)(21 alleles, 9 disorders)

Characterization Study in ProgressCharacterization Study in Progress::

Cystic FibrosisCystic Fibrosis –– 12 alleles12 alleles

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RMsRMs for for Huntington Disease TestingHuntington Disease Testing

RM needs for Huntington disease testing were identified by consultation with clinical laboratory directors

Selected 14 pre-existing cell lines from available Coriell. Chose alleles to represent:

Important diagnostic cutoffsMaterials useful for technical reasonsA large range of allele sizes and combinations

Cell culture and DNA preparation – Coriell

Studies in 10 clinical labs + NIST (coordinated by Sue Richards, OHSU)

Manuscript submitted, materials are available from Coriell

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Summary of HD Confirmation Protocol

Each lab received 50μg aliquots from the 14 HD lines Each sample was tested in 3 separate runs using their in-house assayLabs sent all data to Sue Richards, who

Examined data qualityChecked for discrepanciesCompiled and anonymize data, forward to CDC

NIST – DNA sequence analysisRepeat sizes - expressed as an estimate with a 95% confidence interval

1099 (95.8-102.7)22 (20.3-23.3)NA20253

74 (72.0-76.6)17 (15.4-18.2)NA20210

66 (63.7-67.5)22 (21.2-22.9)NA20252

50 (49.1-50.8)39 (38.1-40.0)NA20251

47 (46.3-47.8)45 (43.8-46.2)NA20209

45 (43.5-46.5)35 (33.4-36.3)NA20208

40 (39.2-41.0)15 (14.1-15.8)NA20250

39 (38.3-39.9)22 (21.1-22.8)NA20249

36 (35.1-37.3)17 (15.0-19.3)NA20248

29 (28.1-29.9)15 (14.1-15.9)NA20247

24 (22.3-25.9)15 (14.1-15.9)NA20246

21 (20-22.1) 19 (17.7-20.7)NA20207

18 (17.2-19.1)17 (15.9-18.3)NA20206

15 (13.7-15.8)15 (13.7-15.8)NA20245

Mean CAG Repeat LengthCoriell Cell Line # Allele 1 (95% CI) Allele 2 (95% CI)

Characterized Huntington Disease Reference MaterialsCharacterized Huntington Disease Reference Materials

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Reference Materials for Reference Materials for Fragile XFragile X

Fragile X workgroup identified fragile X RM Fragile X workgroup identified fragile X RM needsneeds

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

Cell culture and DNA preparationCell culture and DNA preparation-- CoriellCoriell

Characterization by 9 labs using both inCharacterization by 9 labs using both in--house house and Celera assay + DNA sequencing (NIST) and Celera assay + DNA sequencing (NIST) (Fragile X(Fragile X--pertsperts + AMP, + AMP, Jean AmosJean Amos--Wilson, Wilson, Coordinator)Coordinator)

Manuscript completed, DNA available soonManuscript completed, DNA available soon

1220/ no consensusFNA20239

29/ no consensusFNA20241

No consensusMNA20237

30/73FNA20242

117 (only on common platform)MNA20233

30/80FNA20240

76MNA20231

31/53FNA20236

53MNA20230

31/46FNA20234

46MNA20232

29/45FNA20235

29/41FNA20243

41MNA20244

29/30FNA20238

29/29FNA07538

Consensus LengthGenderCell Line

Characterized Fragile X Characterized Fragile X RMsRMs

120 120

SubmitterSubmitterestimateestimate

30/12030/12023/20023/200

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RMsRMs forforAshkenazi Jewish PanelAshkenazi Jewish Panel

RM material needs for Ashkenazi Jewish testing RM material needs for Ashkenazi Jewish testing were identified by examination of current testing were identified by examination of current testing panelspanels

Cell culture and DNA preparation from 27 cell Cell culture and DNA preparation from 27 cell lines (9 disorders) lines (9 disorders) -- CoriellCoriell

Studies were completed by 6 labs using 3 Studies were completed by 6 labs using 3 different platforms/assays different platforms/assays (coordinated by (coordinated by Kasinathan MuralidharanKasinathan Muralidharan))

21 mutations identified 21 mutations identified

The materials are available from The materials are available from CoriellCoriell!!

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TayTay--sachssachs diseasedisease: G269S, 1278insTATC, : G269S, 1278insTATC, IVS12(+1)G>C IVS12(+1)G>C CanavanCanavan diseasedisease: E285A, Y231X : E285A, Y231X Familial Familial dysautonomiadysautonomia: 2507(+6)T>C : 2507(+6)T>C MucolipidosisMucolipidosis IVIV: IVS3(: IVS3(--2)A>G, del6.4kb 2)A>G, del6.4kb NiemannNiemann--Pick disease type APick disease type A: R608del, L302P, : R608del, L302P, P330fs, R496L P330fs, R496L FanconiFanconi anemia group Canemia group C: 322delG, IVS4(+4)A>T : 322delG, IVS4(+4)A>T Bloom syndromeBloom syndrome: 2281del6ins7 : 2281del6ins7 GaucherGaucher diseasedisease: N370S, L444P, 84G>GG, V394L: N370S, L444P, 84G>GG, V394LGlycogen storage disease 1aGlycogen storage disease 1a: Q347X, R83C : Q347X, R83C

Alleles Confirmed in AJ Panel Alleles Confirmed in AJ Panel RMsRMs

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Cystic Fibrosis Cystic Fibrosis –– In progress!In progress!

11 DNA samples11 DNA samples

12 CF alleles (6 new to 12 CF alleles (6 new to CoriellCoriell))

7 laboratories, 5 different platforms/assays + 7 laboratories, 5 different platforms/assays + sequencingsequencing

Labs Coordinated by Vicky Pratt, Quest DiagnosticsLabs Coordinated by Vicky Pratt, Quest Diagnostics

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Cystic Fibrosis

Upon completion of the current study, we will have confirmed by assay and/or DNA sequence analysis 48 different CF alleles (all from Coriell cell lines), including the 23 alleles recommended by the ACMG.

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Development of New Development of New RMsRMs

2 Methods2 Methods

1.1. Establish new cell linesEstablish new cell lines

2. Identify mutations in existing cell lines2. Identify mutations in existing cell lines

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1. Establishing New Cell Lines1. Establishing New Cell Lines

Submission of residual blood from clinical labsSubmission of residual blood from clinical labs4 labs (Quest VA+CA, 4 labs (Quest VA+CA, ViromedViromed, Specialty , Specialty Labs) have sent 41 residual blood to Labs) have sent 41 residual blood to CoriellCoriellSamples represent 11 disordersSamples represent 11 disorders

Collection of blood from patientsCollection of blood from patientsPresented talk at Genetic Alliance annual Presented talk at Genetic Alliance annual meetingmeeting-- have support have support Will approach patient groups for donationsWill approach patient groups for donations

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2. Identify mutations in existing 2. Identify mutations in existing cell linescell lines

Worked with Worked with CoriellCoriell to identify a number to identify a number of preof pre--existing cell lines containing existing cell lines containing unidentified mutations for diseases on our unidentified mutations for diseases on our reference material wish listreference material wish list

DNA was prepared from these lines and DNA was prepared from these lines and sent to volunteer labs for mutation sent to volunteer labs for mutation identificationidentification

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WhatWhat’’s in the works???s in the works???

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RMsRMs to be confirmed to be confirmed ––short termshort term

12 + 3 12 + 3 cystic fibrosiscystic fibrosis allelesalleles1 (possibly 2) 1 (possibly 2) MTHFRMTHFR genotypesgenotypes1 1 GaucherGaucher alleleallele4 4 AlphaAlpha--11--AntitrypsinAntitrypsin genotypesgenotypes1 1 MCADMCAD allele (the major one)allele (the major one)7 7 GalactosemiaGalactosemia allelesalleles1 1 MSUD MSUD alleleallele2 2 MEN2MEN2 allelesalleles2 2 BRCA1BRCA1 allelesalleles1 1 BRCA2BRCA2 alleleallele4 4 CAHCAH allelesalleles

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RMsRMs to be confirmed to be confirmed ––short termshort term-- StatusStatus

DNA is being prepared by DNA is being prepared by CoriellCoriell

I will be rounding up volunteer laboratories for I will be rounding up volunteer laboratories for allele confirmation studiesallele confirmation studies……....

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RMsRMs to be confirmed to be confirmed ––Longer termLonger term

New cell linesNew cell lines

Existing cell lines with needed alleles that are Existing cell lines with needed alleles that are identified by preliminary testingidentified by preliminary testing

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Data CollectionData Collection

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PharmacogeneticPharmacogenetic locilociI collected data from a number of research studies, I collected data from a number of research studies, clinical labs and manufacturers that had genotyped clinical labs and manufacturers that had genotyped hundreds of preexisting hundreds of preexisting CoriellCoriell cell lines for cell lines for pharmacogeneticpharmacogenetic lociloci

Info sources:Info sources:Dr. HDr. H--Y Yuan, Academia Y Yuan, Academia Sinica,TiawanSinica,TiawanDr. M. Dr. M. ReiderReider, U. Washington, U. WashingtonARUP LaboratoriesARUP LaboratoriesChildrenChildren’’s Mercy Hospital, Kansas City, MOs Mercy Hospital, Kansas City, MORoche Molecular SystemsRoche Molecular SystemsThird Wave TechnologiesThird Wave TechnologiesTM BiosciencesTM Biosciences

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PharmacogeneticPharmacogenetic lociloci

Prepared tables that list cell line #, alleles, genotypes Prepared tables that list cell line #, alleles, genotypes and assay methods. and assay methods.

Tables posted on Tables posted on GeTGeT--RM website:RM website:VKORC1VKORC1VKORC1 and CYP2C9VKORC1 and CYP2C9UGT1A1UGT1A1CYP2D6CYP2D6CYP2C9CYP2C9CYP2C19CYP2C19CYP2D6 and/or CYP2C9 and/or CYP2C19CYP2D6 and/or CYP2C9 and/or CYP2C19

The website also lists commercially available productsThe website also lists commercially available products

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Example PGX TableExample PGX Table

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RMsRMs for other disordersfor other disorders(what is on the plate next??)(what is on the plate next??)

??

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RMsRMs for other disorders for other disorders (what is on the plate next??)(what is on the plate next??)

Expanded Newborn Screening!!!Expanded Newborn Screening!!!

In 2005 the American College of Medical In 2005 the American College of Medical Genetics recommended biochemical newborn Genetics recommended biochemical newborn screening for 29 disorders with an additional 25 screening for 29 disorders with an additional 25 disorders that will be identified during the disorders that will be identified during the screening process. screening process.

I am identifying I am identifying CoriellCoriell cell lines that could serve cell lines that could serve as reference materials for genetic confirmation as reference materials for genetic confirmation studies. I hope to have the mutations confirmed studies. I hope to have the mutations confirmed by DNA sequence analysis.by DNA sequence analysis.

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RMsRMs for other disorders for other disorders (what is on the plate next??)(what is on the plate next??)

Molecular OncologyMolecular Oncology

Infectious DiseaseInfectious Disease

Biochemical Genetic TestsBiochemical Genetic Tests

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GeTGeT--RMRM::http://www.phppo.cdc.gov/dls/genetics/qcmaterials/default.aspxhttp://www.phppo.cdc.gov/dls/genetics/qcmaterials/default.aspx