Accurate diagnosis of spinal muscular atrophy and 22q11.2 deletion syndrome using limited deoxynucleotide triphosphates and high-resolution melting

Abstract

BackgroundCopy number variation (CNV) has been implicated in the genetics of multiple human diseases. Spinal muscular atrophy (SMA) and 22q11.2 deletion syndrome (22q11.2DS) are two of the most common diseases which are caused by DNA copy number variations. Genetic diagnostics for these conditions would be enhanced by more accurate and efficient methods to detect the relevant CNVs.MethodsCompetitive PCR with limited deoxynucleotide triphosphates (dNTPs) and high-resolution melting (HRM) analysis was used to detect 22q11.2DS, SMA and SMA carrier status. For SMA, we focused on the copy number of SMN1 gene. For 22q11.2DS, we analyzed CNV for 3 genes (CLTCL1, KLHL22, and PI4KA) which are located between different region-specific low copy repeats. CFTR was used as internal reference gene for all targets. Short PCR products with separated Tms were designed by uMelt software.ResultsOne hundred three clinical patient samples were pretested for possible SMN1 CNV, including carrier status, using multiplex ligation-dependent probe amplification (MLPA) commercial kit as gold standard. Ninety-nine samples consisting of 56 wild-type and 43 22q11.2DS samples were analyzed for CLTCL1, KLHL22, and PI4KA CNV also using MLPA. These samples were blinded and re-analyzed for the same CNVs using the limited dNTPs PCR with HRM analysis and the results were completely consistent with MLPA.ConclusionsLimited dNTPs PCR with HRM analysis is an accurate method for detecting SMN1 and 22q11.2 CNVs. This method can be used quickly, reliably, and economically in large population screening for these diseases.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4833-4) contains supplementary material, which is available to authorized users.

Detection of PTCH1 Copy-Number Variants in Mosaic Basal Cell Nevus Syndrome

“…Thw=e limitation of MLPA in the establishment of a genetic diagnosis in mosaic patients was among others illustrated by a case study on clinically unaffected parents of patients with tuberous sclerosis complex and neurofibromatosis type 1, where it was shown that MLPA was also less sensitive than FISH or PCR for detecting large rearrangements [16,17]. In diagnostic settings, but also in most of the reported MLPA studies, a theoretical or arbitrary ratio range is commonly used, which is uniform for all probes in the mix, and defines the cut-off value with respect to the normal range (e.g., 0.75-1.25, 0.8-1.2, or 0.95-1.05) [16,[18][19][20]. As illustrated in our case study, such a "safe" arbitrary ratio range could easily result in missing a CNV.…”

Current Advances in Genetic Testing for Spinal Muscular Atrophy

“…1 ). Quantitative PCR-based approaches are more suitable as first-tier testing tools for patient diagnosis and newborn screening [ 9 , 10 , 27 , 51 ] than carrier screening and SMN2 copy number determination [ 54 , 56 ]. The primary factor is the inherent challenge of precisely discerning various copy number combinations of SMN genes using non-absolute quantitative methods [ 12 , 27 , 51 , 56 ].…”