2.1. Study Population and DNA Samples

In the present study, 129 blood samples were included. Of these, 54 samples had their SMN1/SMN2 genotype previously obtained by Multiplex ligation-dependent probe amplification (MLPA) and recorded at our center (hereafter referred to as retrospective cohort). The other 75 samples were recruited through convenience sampling (hereafter referred to as prospective cohort) and included blood samples collected from SMA and non-SMA patients (20 with a clinical hypothesis of SMA, 40 SMA relatives and possible carriers, and 15 non related and non-SMA individuals). The DNA samples were extracted from peripheral blood collected in a 4-mL tube containing EDTA through Autopure LS (Qiagen/Gentra, Hilden, Germany) (retrospective cohort) or Biorobot QIAsymphony (Qiagen, Hilden, Germany) (prospective cohort), according to the manufacturer’s protocols, and stored at −20 °C. Additionally, individuals either from the retrospective or prospective cohort (n = 26) were randomly assigned to also have their blood collected on S&S 903 filter paper (kindly supplied by Instituto Jô Clemente, São Paulo, Brazil) for the sample source validation. These samples will be hereafter referred to as the mixed cohort (Supplementary Table S1). Dried blood spots (DBS) were stored in low-permeability zip-lock bags with silica gel desiccant packs at 4 °C (up to six months) and at −20 °C if longer prior to DNA extraction. The DNA was obtained from DBS, as described in Strunk et al. [32]. Briefly, a single 3.2-mm punch was incubated at room temperature for 15 min in 100 uL of 10 mM of NaOH. After, the liquid was washed out, and the procedure was repeated; the punch was incubated at 99 °C for 15 min in 50 uL of 10-mM NaOH. All extracted DNA samples could be stored at −20 °C, but the use of freshly non-diluted DNA was recommended. Three 3.2-mm discs were punched from a blank paper filter between each sample to avoid cross-contamination.

The study was approved by the Ethics Committee in Research (ECR)—Human Beings of the Institute of Bioscience at USP, São Paulo, Brazil (CAAE: 86638318.2.0000.5464). All the patients, parents, and guardians provided signed informed consent accordingly.