抽象的

Unconventional droplet digital PCR assays for SMN1/SMN2 assignment of pathogenic point mutations.

Michela Bulfoni1*, Elena Betto2, Lorenzo Verriello3, Maria Elena Pessa3, Daniela Cesselli1,4, Francesco Curcio1,2 & Incoronata Lonigro1,2

Almost 4% of spinal muscular atrophy affected patients (SMA) carry the deletion in one copy of the Survival Motor Neuron gene 1 (SMN1) and a deleterious point mutation on the other copy of the gene. Correct assignment of the point mutations to the disease causative gene is not a rapid procedure due to the existence of the twin Survival Motor Neuron gene 2 (SMN2) not involved in the onset of the disease. For many years, transcripts sequence, cloning and clone screening were the standard procedures for establishing the unequivocal localization of a point mutation within SMN1 or SMN2 allowing proper diagnosis for symptomatic subjects and carrier testing for relatives at reproductive risk. Many clinical diagnostic laboratories have recently adopted long-range PCR and Sanger sequencing for the identification of the deleterious point mutations, splicing variants and small indels in specifically SMN1. However, the techniques mentioned above are somewhat time-consuming, while great benefits can derive for children with SMA from an early diagnosis and an early entry into a therapeutic trial. The method we developed relay on ddPCR technology and gene- and SNP-specific TaqMan MGB probes to simplify experimental procedures and reduce the turnaround time to reach the correct diagnosis. In addition, this method has the potential to have a more general application in detecting mono or bi-allelic segregation of deleterious coding point mutations in many other recessive inherited diseases, such as some of the results obtained through NGS based tests, without the need to analyze parents or other relatives of the patient.