Abstract
Background: Myotonic dystrophy type 1 (DM1) is caused by an expanded CTG repeat in the non-coding 3’ UTR of the DMPK gene. PCR and Southern Blot Analysis (SBA) of long-range PCR represent the routine molecular testing most widely used for DM1 diagnosis. However, in these conventional methods artifacts such as allele dropout (ADO) represent a risk of misdiagnosis for DM1. Subjects, who show a single product by conventional methods, require a complementary technique such as triplet repeat primed PCR (TP-PCR). Object: To estimate and minimize the incidence of allele dropout event in our diagnostic molecular laboratory by the use of new kit TP-PCR-based. Methods: We retrospectively studied 190 DMPK alleles, on blood samples from to ninety-five subjects, previously genotyped by traditional methods to validate a new assay. The pedigree of a DM1 family was used to expand our analysis. Results: TP-PCR assay correctly identified all 95/95 (100%) subjects and these results were in agreement with the other molecular laboratory. By conventional methods the amplification failure due to allele dropout in our cohort was in 12/190 (6.3%) DMPK alleles analyzed. When these 12 alleles were detected and solved by new assay, we found that the 2.6% was caused by primer sequence-dependent and the remaining 3.6% by polymerase-hindering secondary structures. Conclusions: Allele dropout could be considered as a potentially important problem in DM1 diagnosis that may lead to the attribution of a wrong genotype with long-term consequences for both proband and family.
| Original language | English |
|---|---|
| Pages (from-to) | 111-116 |
| Number of pages | 6 |
| Journal | Clinica Chimica Acta |
| Volume | 484 |
| DOIs | |
| Publication status | Published - Sep 1 2018 |
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Keywords
- Diagnostic error
- Interruptions
- Myotonic dystrophy type 1
- TP-PCR technique
ASJC Scopus subject areas
- Biochemistry
- Clinical Biochemistry
- Biochemistry, medical
Cite this
Incidence of amplification failure in DMPK allele due to allelic dropout event in a diagnostic laboratory. / De Siena, Claudia; Cardani, Rosanna; Brigonzi, Elisa; Bosè, Francesca; Fossati, Barbara; Meola, Giovanni; Costa, Elena; Valaperta, Rea.
In: Clinica Chimica Acta, Vol. 484, 01.09.2018, p. 111-116.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Incidence of amplification failure in DMPK allele due to allelic dropout event in a diagnostic laboratory
AU - De Siena, Claudia
AU - Cardani, Rosanna
AU - Brigonzi, Elisa
AU - Bosè, Francesca
AU - Fossati, Barbara
AU - Meola, Giovanni
AU - Costa, Elena
AU - Valaperta, Rea
PY - 2018/9/1
Y1 - 2018/9/1
N2 - Background: Myotonic dystrophy type 1 (DM1) is caused by an expanded CTG repeat in the non-coding 3’ UTR of the DMPK gene. PCR and Southern Blot Analysis (SBA) of long-range PCR represent the routine molecular testing most widely used for DM1 diagnosis. However, in these conventional methods artifacts such as allele dropout (ADO) represent a risk of misdiagnosis for DM1. Subjects, who show a single product by conventional methods, require a complementary technique such as triplet repeat primed PCR (TP-PCR). Object: To estimate and minimize the incidence of allele dropout event in our diagnostic molecular laboratory by the use of new kit TP-PCR-based. Methods: We retrospectively studied 190 DMPK alleles, on blood samples from to ninety-five subjects, previously genotyped by traditional methods to validate a new assay. The pedigree of a DM1 family was used to expand our analysis. Results: TP-PCR assay correctly identified all 95/95 (100%) subjects and these results were in agreement with the other molecular laboratory. By conventional methods the amplification failure due to allele dropout in our cohort was in 12/190 (6.3%) DMPK alleles analyzed. When these 12 alleles were detected and solved by new assay, we found that the 2.6% was caused by primer sequence-dependent and the remaining 3.6% by polymerase-hindering secondary structures. Conclusions: Allele dropout could be considered as a potentially important problem in DM1 diagnosis that may lead to the attribution of a wrong genotype with long-term consequences for both proband and family.
AB - Background: Myotonic dystrophy type 1 (DM1) is caused by an expanded CTG repeat in the non-coding 3’ UTR of the DMPK gene. PCR and Southern Blot Analysis (SBA) of long-range PCR represent the routine molecular testing most widely used for DM1 diagnosis. However, in these conventional methods artifacts such as allele dropout (ADO) represent a risk of misdiagnosis for DM1. Subjects, who show a single product by conventional methods, require a complementary technique such as triplet repeat primed PCR (TP-PCR). Object: To estimate and minimize the incidence of allele dropout event in our diagnostic molecular laboratory by the use of new kit TP-PCR-based. Methods: We retrospectively studied 190 DMPK alleles, on blood samples from to ninety-five subjects, previously genotyped by traditional methods to validate a new assay. The pedigree of a DM1 family was used to expand our analysis. Results: TP-PCR assay correctly identified all 95/95 (100%) subjects and these results were in agreement with the other molecular laboratory. By conventional methods the amplification failure due to allele dropout in our cohort was in 12/190 (6.3%) DMPK alleles analyzed. When these 12 alleles were detected and solved by new assay, we found that the 2.6% was caused by primer sequence-dependent and the remaining 3.6% by polymerase-hindering secondary structures. Conclusions: Allele dropout could be considered as a potentially important problem in DM1 diagnosis that may lead to the attribution of a wrong genotype with long-term consequences for both proband and family.
KW - Diagnostic error
KW - Interruptions
KW - Myotonic dystrophy type 1
KW - TP-PCR technique
UR - http://www.scopus.com/inward/record.url?scp=85047636906&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85047636906&partnerID=8YFLogxK
U2 - 10.1016/j.cca.2018.05.040
DO - 10.1016/j.cca.2018.05.040
M3 - Article
C2 - 29803895
AN - SCOPUS:85047636906
VL - 484
SP - 111
EP - 116
JO - Clinica Chimica Acta
JF - Clinica Chimica Acta
SN - 0009-8981
ER -