Incidence of amplification failure in DMPK allele due to allelic dropout event in a diagnostic laboratory.

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.

Cardiac involvement in myotonic dystrophy: The role of troponins and N-terminal pro B-type natriuretic peptide.

BACKGROUND AND AIMS: Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are dominant inherited muscular dystrophies with multiple systemic involvement, often producing cardiac injury. This study sought to determine the clinical significance of elevated high sensitivity cardiac troponin T and I (hs-cTnT and hs-cTnI), and N-terminal pro B-type natriuretic peptide (NT-pro-BNP) in this population. METHODS: Sixty DM patients (35 men and 25 women; mean age: 45.1 years, range: 12-73 years) underwent clinical cardiac investigations and measurements of serum hs-cTnT, hs-cTnI, creatine kinase (CK), and NT-proBNP. Left ventricular (LV) ejection fraction (EF) was assessed by echocardiography. RESULTS: Genetic analysis revealed that 46 of the 60 patients were DM1, and 14 DM2. Blood measurements showed persistent elevation of hs-cTnT and CK in 55/60 DM patients (91.73%). In contrast, hs-cTnI values were persistently normal throughout the study. Only 2 patients showed an EF <50%, being the overall range of this population between 40% and 79%. We found ECG abnormalities in 19 patients. Of these patients, 13 showed first or second-degree atrio ventricular (AV) blocks (PR interval ≥ 200 ms), 4 showed a left bundle branch block (LBBB) prolonged (QRS duration ≥120 ms), and 2 had an incomplete bundle branch block (QRS duration between 110 and 119 ms). After excluding patients with EF <50%, NT-pro-BNP measurement > 125 pg/mL was an independent predictor of ECG abnormalities. CONCLUSIONS: NT-pro-BNP levels may be considered to be used clinically to identify DM patients at increased risk of developing myocardial conduction abnormalities.

High-sensitive cardiac troponin T (hs-cTnT) assay as serum biomarker to predict cardiac risk in myotonic dystrophy: A case-control study.

BACKGROUND: Myotonic dystrophy (DM) is a genetic disorder caused by nucleotide repeats expansion. Sudden death represents the main cause of mortality in DM patients. Here, we investigated the relationship between serum cardiac biomarkers with clinical parameters in DM patients. METHODS: Case-control study included 59 DM patients and 22 healthy controls. An additional group of 62 controls with similar cardiac defects to DM were enrolled. RESULTS: NT-proBNP, hs-cTnT and CK levels were significantly increased in DM patients compared to healthy subjects (p=0.0008, p<0.0001, p<0.0001). Also, hs-cTnT levels were significantly higher in DM compared to control group with cardiac defects (p=0.0003). Positive correlation was found between hs-cTnT and hs-cTnI in both DM patients and controls (p=0.019, p=0.002). Independently from the age, the risk of DM disease was positively related to an increase in hs-cTnT (p=0.03). On the contrary, the risk of DM was not related to hs-cTnI, but was evidenced a role of PR interval (p=0.03) and CK (p=0.08). CONCLUSIONS: The levels of hs-cTnT were significantly higher in DM patients. Analysis, with anti-cTnT, shows that this increase might be linked to heart problems. This last finding suggests that hs-cTnT might represent a helpful serum biomarker to "predict" cardiac risk in DM disease.