Identification of deletion and duplication genotypes of thePMP22gene using PCR‐RFLP, competitive multiplex PCR, and multiplex ligation‐dependent probe amplification: A comparison

Abstract

We evaluated the efficacy of PCR-RFLP, competitive multiplex PCR, and a commercially available system of multiplex ligation-dependent probe amplification (MLPA) for the determination of deletion and duplication genotypes of the PMP22 gene. We compared the methods for efficiency, sensitivity, and specificity. We determined the gene dosage of the PMP22 gene via PCR-RFLP, competitive multiplex PCR, and MLPA. To demonstrate the sensitivity and accuracy of these three methods, a total of 185 samples from 42 patients with hereditary neuropathy with liability to pressure palsies (HNPP), 57 patients with Charcot-Marie-Tooth disease type 1A (CMT1A), and 86 unaffected individuals, were analyzed. Molecular diagnosis by PCR-RFLP was performed on all 185 samples; 24 HNPP deletions and 33 CMT1A duplications were identified. In contrast, 25 HNPP deletions and 38 CMT1A duplications were identified correctly using competitive multiplex PCR and MLPA. Six samples were incorrectly identified by PCR-RFLP (one HNPP deletion and five CMT1A duplications). Competitive multiplex PCR and MLPA demonstrated reliability and relative speed compared to PCR-RFLP; they were superior to PCR-RFLP for gene dosage quantification. Multiplex PCR and MLPA should be the methods of choice for detection of deletion and duplication genotypes in molecular genetic diagnoses.

Reevaluation of the CMT1A duplication frequency in Greek Charcot‐Marie‐Tooth type 1 patients

“…In familial cases the frequency was 66.6% (52/78) and in non-familial cases 30% (15/50). Subsequent screening of the 128 cases with long PCR revealed an overall sensitivity of 67.16%, lower than figures quoted in the literature (84-87%) (2,4). In familial cases the sensitivity was lower (59.6%) compared to non-familial cases (93.3%, p = 0.014).…”

Mutational analysis of PMP22, GJB1 and MPZ in Greek Charcot-Marie-Tooth type 1 neuropathy patients

“…It is important to note that the methodology used to detect the CMT1A duplication (long PCR method) has an estimated sensitivity of 84–87% (8, 9). Although this may lead to an underestimation of the duplication frequency, the overall impact is likely to be small and should not alter our main conclusion, placing Greece in the countries with low relative frequency of CMT1A.…”

Mutation spectrum of the fibrillin‐1 (FBN1) gene in Taiwanese patients with Marfan syndrome

“…Faivre et al (2009) recently reported that exons 24-32 represent a hotspot for neonatal MFS and severe forms of MFS. The purpose of this study was to evaluate FBN1 mutations in Taiwanese individuals by using denaturing high performance liquid chromatography (DHPLC) and multiplex ligation-dependent amplification (MLPA) (Liu et al, 1997;Matyas et al, 2002;Kosaki et al, 2005;den Dunnen & White, 2006;Howarth et al, 2007;Kozlowski et al, 2008;Hung et al, 2005Hung et al, , 2006Hung et al, , 2007Hung et al, , 2008aHung et al, , 2008bHung et al, , 2009. DHPLC can detect heteroduplexes of denatured and reannealed PCR amplicons, and has a highcapacity and low-costs.…”