TY - JOUR
T1 - Microdissection Genotyping of Archival Fixative Treated Tissue for Gaucher Disease
AU - Mohan, Deepak
AU - Rolston, Raj
AU - Pal, Rekha
AU - Swalsky, Patricia A.
AU - Sasatomi, Eizaburo
AU - Lee, Robert E.
AU - Finkelstein, Sydney D.
PY - 2004/4
Y1 - 2004/4
N2 - The genetic diagnosis of Gaucher disease by molecular methods is complicated by the existence of a highly homologous transcribed pseudogene (96% identity) that is found in close proximity to the true gene on chromosome 1q21. In addition, the pseudogene sequence can mimic disease-causing mutations in the true gene. Selective polymerase chain reaction (PCR) amplification of the true gene can be accomplished in extracted DNA from fresh-frozen samples by designing oligonucleotide primers to hybridize to defined regions that are not present in the pseudogene. This standard molecular approach, which entails amplification of relatively long segments of intact DNA, is not feasible in archival, paraffin-embedded, solid-tissue specimens in which the negative effects of chemical fixation result in DNA strand scission and breakdown of nucleic acid. A novel approach, specifically created for use with archival, fixative-treated tissue specimens, was developed for detection and characterization of common mutations of Gaucher disease. Three separate robust PCR reactions were formulated, 2 for selective amplification of portions of only the true gene exons 2 and 9, with a third reaction targeting exon 10, wherein both the true and pseudogene were coamplified. In the latter, DNA sequencing was used to determine the presence of true and pseudogene allele content in addition to identification of base sequence alterations. This method, requiring a single, 4-μm-thick histologic section, was successfully applied to archival paraffin block tissue specimens that had been in storage for up to 75 years. It was capable of accurately genotyping common Gaucher disease mutations as well as discovering a novel mutation and genetic polymorphism. We recommend our approach when only fixative-treated tissue is available for molecular genotyping.
AB - The genetic diagnosis of Gaucher disease by molecular methods is complicated by the existence of a highly homologous transcribed pseudogene (96% identity) that is found in close proximity to the true gene on chromosome 1q21. In addition, the pseudogene sequence can mimic disease-causing mutations in the true gene. Selective polymerase chain reaction (PCR) amplification of the true gene can be accomplished in extracted DNA from fresh-frozen samples by designing oligonucleotide primers to hybridize to defined regions that are not present in the pseudogene. This standard molecular approach, which entails amplification of relatively long segments of intact DNA, is not feasible in archival, paraffin-embedded, solid-tissue specimens in which the negative effects of chemical fixation result in DNA strand scission and breakdown of nucleic acid. A novel approach, specifically created for use with archival, fixative-treated tissue specimens, was developed for detection and characterization of common mutations of Gaucher disease. Three separate robust PCR reactions were formulated, 2 for selective amplification of portions of only the true gene exons 2 and 9, with a third reaction targeting exon 10, wherein both the true and pseudogene were coamplified. In the latter, DNA sequencing was used to determine the presence of true and pseudogene allele content in addition to identification of base sequence alterations. This method, requiring a single, 4-μm-thick histologic section, was successfully applied to archival paraffin block tissue specimens that had been in storage for up to 75 years. It was capable of accurately genotyping common Gaucher disease mutations as well as discovering a novel mutation and genetic polymorphism. We recommend our approach when only fixative-treated tissue is available for molecular genotyping.
KW - Gaucher's disease
KW - Molecular anatomical pathology (MAP)
KW - Molecular genotyping (MG)
KW - Paraffin
KW - Polymerase chain reaction (PCR)
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U2 - 10.1016/j.humpath.2003.11.006
DO - 10.1016/j.humpath.2003.11.006
M3 - Article
C2 - 15116330
AN - SCOPUS:1942517780
SN - 0046-8177
VL - 35
SP - 482
EP - 487
JO - Human Pathology
JF - Human Pathology
IS - 4
ER -