Patients
Patients, who applied to the Department of Child Health and Diseases and also the Department of Adult Neurology were pre-diagnosed with SMA were referred to the Department of Medical Genetics for genetic testing. A total of 58 patients were determined between 2018 and 2021 diagnosed with SMA according to clinical findings and genetic test results, and those who fulfilled the inclusion criteria were included in the study. 39 children (0-18 years) and 19 adults (18-50 years) a total of 58 patients, 28 children and 12 adult healthy controls were analyzed in terms of NAIP and GTF2H2 gene deletions. The study was conducted both retrospectively by examining the Multiplex Ligation Dependent Probe Amplification (MLPA) results of SMA patients, and prospectively by studying multiplex PCR for the NAIP and GTF2H2 gene with samples taken from these patients and the healthy control group.
The patients included according to the diagnostic criteria for SMA defined by the International SMA Consortium. Patients or their parents were informed about the study and taken a written informed consent for the genetics analysis from all participants. This study was approved by Erciyes University Clinical Research Ethics Committee.
Establishment of healthy control groups:
To the healthy control group; children and adults who applied to the Pediatric Neurology outpatient clinic and Adult Neurology outpatient clinic and who did not have any SMA or other neuromuscular disease findings as a result of the clinical examination, who do not have any chronic disease according to the anamnesis and who do not use regular medication, was included.
Methods
A retrospective examination of SMA patients who had previously genetically analyzed by using the MLPA method:
2cc peripheral blood samples were taken from these patients with an EDTA tube and DNA was extracted. Mutation status in exon 7 and exon 8 regions of SMN1 and SMN2 genes was analyzed with MLPA method according to the manufacturer’s protocol. In this context, the SALSA MLPA P021 kit was used and the MLPA PCR products were loaded on the ABI Prisma 3500 genetic analyzer (Applied Biosystems) with LIZ 500 as the size standard. The results were evaluated in the Coffalyser program and the copy numbers were determined. The files of the patients were investigated who were found to have homozygous deletions in the SMN1 gene.
Prospective molecular analysis of the NAIP gene and the GTF2H2 gene associated with SMA disease:
NAIP gene exon 4 and 5 deletions, were analyzed in 58 patients (39 children + 19 adults) diagnosed with SMA and compared with 40 healthy control. The DNA products that available in the Department of Medical Genetics of pediatric and adult patients who were previously positive for SMA type1 homozygous deletions were used. As the control group, 2 ml of EDTA blood taken from healthy volunteers at the same age as our patients, and DNA was extracted. The DNA quality and concentrations were measured in the nanodrop.
PCR amplification for NAIP gene was performed by primer oligonucleotide sequences of exon 5: 5F, 5'TGCCACTCCCAGGCAATCTAA-3', 5R, 5'-CATTTGGCATGTTCCTTCCAAG-3', and exon 4: 4F, 5’-AAAGCCTCTGACGAGAGGATC-3’ and 4R, 5’-CTCTCAGCCTGCTCTTCAGAT-3’. Also, the primer exon 13: 13F, 5’-ATGCTTGGATCTCTAGAATGG-3’ and 13R, 5’-CCAGCTCCTAGAGAAAGAAGGA-3’ was used as an internal control. Exon 4, 5 and 13 of NAIP were amplified with multiplex PCR. (Liang et al. 2009). Multiplex PCR was performed on Verriti Thermal Cycler. Each reaction was carried out in a 50-μl volume containing 10 mmol/L reaction buffer, 1.5 mmol/L MgCl2, 200 μmol/L each dNTPs, 0.2 μmol/L each primer, approximately 200 ng genomic DNA, and 5U/μl Taq polymerase (Thermo). Multiplex PCR was carried out with an initial denaturation at 94 °C for 6 min, followed by 35 cycles (95 °C, 59 °C, and 72 °C for 1 min each) and a final extension step at 72 °C for 7 min. The PCR products were run for 45 minutes on a 3% agarose gel with 100 volts current. The results were evaluated under ultraviolet (UV) and the presence or absence of exon 4 and 5 of the NAIP gene was verified.
GTF2H2 gene analyzed with RFLP (Restriction Fragment Length Polymorphism) method. PCR amplification for GTF2H2 gene was performed by primer oligonucleotide sequences of exon 10: 10F, 5’-CGTACCATGTTATTTTAGATG-3’ and 10R, 5’-TACGAATAAGTGAGCATTCAG-3’. PCR reaction was carried through with a 50-μl volume in same conditions with the NAIP gene and PCR program was set initial denaturation at 95 °C for 5 min, followed by 36 cycles (94 °C for 40 sec, 50 °C for 45 sec, and 72 °C for 45 sec) and a final extension step at 72 °C for 10 min (Carter et al. 1997). The PCR products were cut using restriction enzymes DdeI and Hinf1II at 37 °C overnight. On a 3 percent agarose gel with 90 volts current, the cut products with the enzyme were run for one hour. The cut and uncut conditions of the GTF2H2 gene were determined under ultraviolet (UV) illumination.
Clinical evaluation
The clinical features of all SMA patients in our study group, including ambulation, muscle weakness, hypotonia and respiratory distress were noted. In addition, an expanded version of the HFMSE was used for the motor function of SMA type 2 and 3 patients. The HFMSE, a scale specifically designed for SMA, is scored from 0 to 66 points, and higher scores indicate higher motor function (O’Hagen et al. 2007)(Pera et al. 2017).
Statistical analyzes
Normality tests were performed in all comparisons. The relationship between various subtypes of SMA and ambulation status was determined by the non-parametric Fisher's exact test (SPSS version 26). To compare clinical subtypes and HMFSE scores, the non-parametric Mann Whitney U test was performed and shown as a bar graph (GraphPad Prism 8). The distribution of SMN2 exon 7 copy number variants (CNVs) by clinical subtypes was determined by Fisher's exact test (SPSS version 26), and categorical data were expressed as percentages. Also, in order to determine where the significance originates from, post-hoc analysis was performed in the comparison of subgroups.