Snapback Primer Genotyping of the Gilbert Syndrome UGT1A1 (TA)n Promoter Polymorphism by High-Resolution Melting

J.S. Farrar, R.A. Palais, and C.T. Wittwer

www.clinchem.org/cgi/content/article/57/9/1303

September 2011

© Copyright 2011 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry

IntroductionIntroduction Gilbert syndrome

A chronic non-hemolytic unconjugated hyperbilirubinemia Associated with thymine-adenine (TA) insertions in the

UGT1A1 promoter UGT1A1 promoter genotype also correlates with toxicity

induced by the chemotherapeutic drug irinotecan

© Copyright 2009 by the American Association for Clinical Chemistry

IntroductionIntroduction UGT1A1 (TA)n promoter polymorphism

Repeat length varies: (TA)5 , (TA)6 , (TA)7 , (TA)8

Frequency and length of repeats depends on ethnicity

(TA)5 and (TA)8 alleles restricted to African descent Current genotyping methods

Multiple manual steps, multiple labeled probes and/or difficulty genotyping the (TA)5 and (TA)8 alleles

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction (cont)Introduction (cont) Snapback primer genotyping

Closed-tube, high-resolution melting method Uses a saturating double-stranded DNA dye instead of

covalently modified primers/probes Only two standard PCR primers are needed (one with a

5’-addition) Has previously been applied to single-base variants and

small deletions

© Copyright 2009 by the American Association for Clinical Chemistry

QuestionQuestion Why is it important that UGT1A1 (TA)n genotyping assays be validated for the (TA)5 and (TA)8 repeat alleles?

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Snapback primer approach for genotyping the UGT1A1 (TA)n promoter polymorphism. A probe complementary to the (TA)5 repeat is added as a 5’ addition to the forward PCR primer (gray). Asymmetric PCR overproduces a single-stranded DNA product that forms an intra-molecular hairpin. Products with repeats greater than (TA)5 form bulge loops that progressively destabilize the hairpin structure. Hairpin stability is revealed by high-resolution melting analysis.

Materials and MethodsMaterials and Methods

© Copyright 2009 by the American Association for Clinical Chemistry

Materials and Methods (cont)Materials and Methods (cont) 100 African American DNA samples

Study population enriched for (TA)5 and (TA)8 alleles

New melting analysis method Plots the local deviation from exponential decay in

order to remove background fluorescence Improves genotype clustering

© Copyright 2009 by the American Association for Clinical Chemistry

Materials and Methods (cont)Materials and Methods (cont) 3 different genotyping methods

Fragment analysis (reference method) Small amplicon melting Snapback primer genotyping

2 blinded studies Small amplicon melting and snapback primer genotyping

on a capillary-based instrument Instrument comparison for snapback primer genotyping

(capillary vs. plate-based)

© Copyright 2009 by the American Association for Clinical Chemistry

QuestionQuestion What structure does a snapback primer form after PCR and how does this structure reveal the (TA)n repeat genotype?

© Copyright 2009 by the American Association for Clinical Chemistry

ResultsResults Snapback primer genotyping

on a capillary-based instrument

99% concordant with genotyping results from fragment analysis

Reanalysis of single discordant sample revealed an error in fragment analysis

100% accuracy after correction for error in fragment

© Copyright 2009 by the American Association for Clinical Chemistry

Results (cont)Results (cont) Snapback primer genotyping on a capillary- based instrument

Although the melting temperature differences are small, the absolute temperature precision of the instrument and the shapes of the melting curves enable accurate genotyping

© Copyright 2009 by the American Association for Clinical Chemistry

Results (cont)Results (cont)

Actual Genotype n Miscalled Genotype n(TA)5/(TA)6 6 ­

(TA)5/(TA)7 7 (TA)5/(TA)6 1

(TA)5/(TA)8 2 ­

(TA)6/(TA)6 22 (TA)5/(TA)6 1

(TA)6/(TA)7 34 (TA)6/(TA)6 4 (TA)7/(TA)7 1

(TA)6/(TA)8 7 (TA)6/(TA)7 3(TA)7/(TA)7 1(TA)7/(TA)8 1

(TA)7/(TA)7 15 (TA)6/(TA)7 1

(TA)7/(TA)8 7 (TA)7/(TA)7 3Total 100 ­ 16

Small amplicon genotyping on a capillary-based instrument

84% accuracy compared to fragment analysis after correction for fragment analysis error

© Copyright 2009 by the American Association for Clinical Chemistry

Results (cont)Results (cont)

 Actual Genotype n Miscalled Genotype n

(TA)5/(TA)6 5 0

(TA)5/(TA)7 6 (TA)6/(TA)7 2

(TA)5/(TA)8 2 (TA)5/(TA)7 1

(TA)6/(TA)6 21 (TA)5/(TA)7 1 (TA)6/(TA)7 1

(TA)6/(TA)7 33 (TA)7/(TA)7 3

(TA)6/(TA)8 7 (TA)6/(TA)7 1(TA)7/(TA)7 3

(TA)7/(TA)7 15 (TA)6/(TA)8 3(TA)7/(TA)8 3

(TA)7/(TA)8 6 0Total 95 ­ 18

Snapback primer genotyping on a plate-based instrument 100% accuracy on

capillary-based instrument fell to 81% on a plate-based instrument

© Copyright 2009 by the American Association for Clinical Chemistry

QuestionQuestion Why is genotyping accuracy dependent on the

instrument?

© Copyright 2009 by the American Association for Clinical Chemistry

ConclusionsConclusions Instrument and genotyping method are critical

for successful genotyping Plate-based high-resolution melting instrument did not

perform as well as capillary-based instrument Absolute temperature precision of the instrument is

critical Snapback primer genotyping performed better than small

amplicon genotyping

Snapback primers can be used to genotype simple sequence repeats in <30 min

© Copyright 2009 by the American Association for Clinical Chemistry

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