Pyrosequencing Technology as a Method for the Diagnosis of Multiple Endocrine Neoplasia Type 2

Kent E. Kruckeberg, Stephen N Thibodeau

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Background: Multiple endocrine neoplasia type 2 (MEN2) is a cancer syndrome with well-characterized causative mutations. Missense mutations in ∼15 codons of the RET gene have been linked to disease phenotypes in the vast majority of cases. These missense mutations range from very simple single nucleotide base changes to more numerous changes at a given codon; they therefore are often tested for by more than one DNA-based diagnostic method. We developed and evaluated a Pyrosequencing™ technology-based approach for MEN2 mutation testing that allows both simple and complex mutations to be analyzed on one platform. Methods: Archived DNA from peripheral blood of patients referred to the Mayo Clinic Molecular Genetics laboratory for MEN2 testing was selected. One to all of codons 609, 611, 618, 620, 630, 634, 768, 804, and 918 were analyzed by Pyrosequencing technology to match the original analysis of each patient. Template PCRs were set up using an automated liquid handler; the subsequent post-PCR preparation step was performed manually, and the sequencing was performed by a PSQ 96 instrument. Samples were tested in batch sizes expected to occur routinely. Results: We analyzed samples from 217 patients who previously tested negative for MEN2 and 230 patients who previously tested positive, for a total of 1449 sequencing reactions. One discrepant result was found (100% concordant for negatives and 99.6% concordant for positives). A total of 37 unique mutations or alterations of unknown significance were analyzed. Conclusion: Pyrosequencing technology offers an accurate, nonisotopic, simple, and rapid method for the analysis of DNA from patients suspected of having MEN2.

Original languageEnglish (US)
Pages (from-to)522-529
Number of pages8
JournalClinical Chemistry
Volume50
Issue number3
DOIs
StatePublished - Mar 2004

Fingerprint

Multiple Endocrine Neoplasia Type 2a
Technology
Codon
DNA
Mutation
Missense Mutation
Testing
Blood
Nucleotides
Genes
Polymerase Chain Reaction
Liquids
Molecular Biology
Phenotype
Neoplasms

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Pyrosequencing Technology as a Method for the Diagnosis of Multiple Endocrine Neoplasia Type 2. / Kruckeberg, Kent E.; Thibodeau, Stephen N.

In: Clinical Chemistry, Vol. 50, No. 3, 03.2004, p. 522-529.

Research output: Contribution to journalArticle

@article{584a70ed9870484da6c1da0aca285d22,
title = "Pyrosequencing Technology as a Method for the Diagnosis of Multiple Endocrine Neoplasia Type 2",
abstract = "Background: Multiple endocrine neoplasia type 2 (MEN2) is a cancer syndrome with well-characterized causative mutations. Missense mutations in ∼15 codons of the RET gene have been linked to disease phenotypes in the vast majority of cases. These missense mutations range from very simple single nucleotide base changes to more numerous changes at a given codon; they therefore are often tested for by more than one DNA-based diagnostic method. We developed and evaluated a Pyrosequencing™ technology-based approach for MEN2 mutation testing that allows both simple and complex mutations to be analyzed on one platform. Methods: Archived DNA from peripheral blood of patients referred to the Mayo Clinic Molecular Genetics laboratory for MEN2 testing was selected. One to all of codons 609, 611, 618, 620, 630, 634, 768, 804, and 918 were analyzed by Pyrosequencing technology to match the original analysis of each patient. Template PCRs were set up using an automated liquid handler; the subsequent post-PCR preparation step was performed manually, and the sequencing was performed by a PSQ 96 instrument. Samples were tested in batch sizes expected to occur routinely. Results: We analyzed samples from 217 patients who previously tested negative for MEN2 and 230 patients who previously tested positive, for a total of 1449 sequencing reactions. One discrepant result was found (100{\%} concordant for negatives and 99.6{\%} concordant for positives). A total of 37 unique mutations or alterations of unknown significance were analyzed. Conclusion: Pyrosequencing technology offers an accurate, nonisotopic, simple, and rapid method for the analysis of DNA from patients suspected of having MEN2.",
author = "Kruckeberg, {Kent E.} and Thibodeau, {Stephen N}",
year = "2004",
month = "3",
doi = "10.1373/clinchem.2003.027128",
language = "English (US)",
volume = "50",
pages = "522--529",
journal = "Clinical Chemistry",
issn = "0009-9147",
publisher = "American Association for Clinical Chemistry Inc.",
number = "3",

}

TY - JOUR

T1 - Pyrosequencing Technology as a Method for the Diagnosis of Multiple Endocrine Neoplasia Type 2

AU - Kruckeberg, Kent E.

AU - Thibodeau, Stephen N

PY - 2004/3

Y1 - 2004/3

N2 - Background: Multiple endocrine neoplasia type 2 (MEN2) is a cancer syndrome with well-characterized causative mutations. Missense mutations in ∼15 codons of the RET gene have been linked to disease phenotypes in the vast majority of cases. These missense mutations range from very simple single nucleotide base changes to more numerous changes at a given codon; they therefore are often tested for by more than one DNA-based diagnostic method. We developed and evaluated a Pyrosequencing™ technology-based approach for MEN2 mutation testing that allows both simple and complex mutations to be analyzed on one platform. Methods: Archived DNA from peripheral blood of patients referred to the Mayo Clinic Molecular Genetics laboratory for MEN2 testing was selected. One to all of codons 609, 611, 618, 620, 630, 634, 768, 804, and 918 were analyzed by Pyrosequencing technology to match the original analysis of each patient. Template PCRs were set up using an automated liquid handler; the subsequent post-PCR preparation step was performed manually, and the sequencing was performed by a PSQ 96 instrument. Samples were tested in batch sizes expected to occur routinely. Results: We analyzed samples from 217 patients who previously tested negative for MEN2 and 230 patients who previously tested positive, for a total of 1449 sequencing reactions. One discrepant result was found (100% concordant for negatives and 99.6% concordant for positives). A total of 37 unique mutations or alterations of unknown significance were analyzed. Conclusion: Pyrosequencing technology offers an accurate, nonisotopic, simple, and rapid method for the analysis of DNA from patients suspected of having MEN2.

AB - Background: Multiple endocrine neoplasia type 2 (MEN2) is a cancer syndrome with well-characterized causative mutations. Missense mutations in ∼15 codons of the RET gene have been linked to disease phenotypes in the vast majority of cases. These missense mutations range from very simple single nucleotide base changes to more numerous changes at a given codon; they therefore are often tested for by more than one DNA-based diagnostic method. We developed and evaluated a Pyrosequencing™ technology-based approach for MEN2 mutation testing that allows both simple and complex mutations to be analyzed on one platform. Methods: Archived DNA from peripheral blood of patients referred to the Mayo Clinic Molecular Genetics laboratory for MEN2 testing was selected. One to all of codons 609, 611, 618, 620, 630, 634, 768, 804, and 918 were analyzed by Pyrosequencing technology to match the original analysis of each patient. Template PCRs were set up using an automated liquid handler; the subsequent post-PCR preparation step was performed manually, and the sequencing was performed by a PSQ 96 instrument. Samples were tested in batch sizes expected to occur routinely. Results: We analyzed samples from 217 patients who previously tested negative for MEN2 and 230 patients who previously tested positive, for a total of 1449 sequencing reactions. One discrepant result was found (100% concordant for negatives and 99.6% concordant for positives). A total of 37 unique mutations or alterations of unknown significance were analyzed. Conclusion: Pyrosequencing technology offers an accurate, nonisotopic, simple, and rapid method for the analysis of DNA from patients suspected of having MEN2.

UR - http://www.scopus.com/inward/record.url?scp=1542358908&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1542358908&partnerID=8YFLogxK

U2 - 10.1373/clinchem.2003.027128

DO - 10.1373/clinchem.2003.027128

M3 - Article

C2 - 14718397

AN - SCOPUS:1542358908

VL - 50

SP - 522

EP - 529

JO - Clinical Chemistry

JF - Clinical Chemistry

SN - 0009-9147

IS - 3

ER -