Applicability of digital PCR to the investigation of pediatric-onset genetic disorders

Abstract

Early-onset rare diseases have a strong impact on child healthcare even though the incidence of each of these diseases is relatively low. In order to better manage the care of these children, it is imperative to quickly diagnose the molecular bases for these disorders as well as to develop technologies with prognostic potential. Digital PCR (dPCR) is well suited for this role by providing an absolute quantification of the target DNA within a sample. This review illustrates how dPCR can be used to identify genes associated with pediatric-onset disorders, to identify copy number status of important disease-causing genes and variants and to quantify modifier genes. It is also a powerful technology to track changes in genomic biomarkers with disease progression. Based on its capability to accurately and reliably detect genomic alterations with high sensitivity and a large dynamic detection range, dPCR has the potential to become the tool of choice for the verification of pediatric disease-associated mutations identified by next generation sequencing, copy number determination and noninvasive prenatal screening.

Identification of the Novel Methylated Genes’ Signature to Predict Prognosis in INRG High-Risk Neuroblastomas

“…Meanwhile, MIMT1 is an MER1 repeat-containing imprinted transcript, which can undergo hypermethylation in the placenta of intrauterine growth-restricted fetuses in cattle [ 51 ], and truncation of exons 3 and 4 of the MIMT1 gene caused intrauterine growth restriction [ 52 ]. Furthermore, the transmembrane p24 trafficking protein family member, DDX28, was used to investigate pediatric-onset genetic disorders by digital PCR [ 53 ]. However, the biological roles of the two genes (TMED and LOC151174) in cancer are yet unknown, and this has to be researched further in future research.…”

Establishing a reference dataset for the authentication of spinal muscular atrophy cell lines using STR profiling and digital PCR

“…Because of this inverse relationship, SMN2 copy number is being used as a criterion for inclusion in SMA clinical trials. dPCR can accurately measure copy number variations found in many pediatric-onset disorders including SMA [41]. We and others have demonstrated that dPCR provides an accurate and reliable measurement of SMN2 copy number [21;42].…”

Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument

“…The Absolute Q dPCR quadruplex assay approach could be extended to identification of copy number variations associated with other diseases, in addition to SMA. dPCR has been used to identify rare gene variants as well as differences in copy number of multiple genes associated with pediatric-onset disorders (reviewed in 24 ). We have demonstrated that this system can identify a rare single nucleotide variant ( EGFR(T790M) ) associated with non-small cell lung carcinoma as well as fusion genes ( BCR-ABL1 and CCDC88C-FLT3 ) that are associated with different types of cancer 29 .…”

“…Digital PCR (dPCR) alleviates the limitations previously noted to provide absolute quantification of a target gene within a sample for molecular diagnostics and prognostics 24 . With dPCR, the prepared PCR sample is distributed across a large number of physically isolated micro-reaction partitions so that each partition will have single digit counts of template DNA or none at all 25 , 26 .…”