الفهرس 
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Abstract
SMA is a collection of neuromuscular ailments Distinguished by the progressive loss of alpha motor neurons in the spinal cord, resulting in gradual muscle atrophy, weakness, and paralysis. The survival motor neuron 1 (SMN1) gene, which is located on the 5q11.2-q13 subchromosome, is the most frequently affected by SMA defects. People with less severe types of SMA have more copies of the SMN2 gene than do patients with more severe forms of the disease. The neighboring gene, neuronal apoptosis inhibitory (NAIP) gene that located on the same chromosomal region is the other candidate gene. The present study aimed to study of Spinal Muscle Atrophy (SMA) disease (type I and II) in pediatric patients in Egypt at biochemical and molecular levels.
This study included the following groups: group I: Sixty-five healthy control of matched age, according to the instruction of the Ethical Committee in Medical Research Institute, a signed consent collected from all the participants in the study. group II: Sixty-five pediatric patients with SMA type I includes 30 patients and Type II includes 35 patients, the patients chosen from patients who admitted to Pediatric Neurology Department in Alexandria Pediatric Hospital, the inclusion criteria of the patients based on clinical assessment and diagnostic investigations, which may include tests such as EMG and nerve conduction velocity studies. from all participants, a blood sample (4 ml) were withdrawn, then it were divided into two tubes, one plain tube and one EDTA-coated tube, for serum and whole blood, respectively. All samples were stored at -20°C until the time of investigations.1- Colorimetric determination of serum creatine Kinase (CK) 2-Determination of the expression of SMN1 and SMN2 genes by transcriptase PCR (qRT-PCR) 3-Determination of the Genetic polymorphisms of SMN1, SMN2 and NAIP genes by RFLP-PCR technique confirmed by DNA sequencing to detect single nucleotide polymorphism (SNP) in some samples. There was no significant difference in serum cpk levels between patients and control. This study showed that, regardless of disease severity, 65 of the total 65 patients (100%) have homozygous absence of SMN1 exons 7. 90% (27/30) SMA1 patients and 88% (31/35) of SMA2 patients showed homozygous deletions for exon 7 and exon 8. This result was in agreement with several previous investigations reporting a higher incidence of this genotype among type I SMA patients as compared to patients with type II All NAIP deleted patients reported in this study lacked also the SMN1 exon 7 or exon 7 and 8.. Homozygous deletions of both exons 7, 8 in SMN1 and exon 5 were seen in 53.3% of type I SMA and 22.9% of type II patient. In the present study, homozygous absence of SMN1 exons 7 & 8 in association with deletion of NAIP exon 5 has been found in most type I patients (16/30; 53.3%) and in smaller percents in type II (8/35; 22.9%) SMA patients. No homozygous deletions were seen in the controls. The findings suggest that the deletion of exon 5 in NAIP could be involved in the molecular mechanisms underlying spinal muscular atrophy (SMA). Furthermore, all patients with SMA types I and II had a copy number distribution of 0 for the SMN1 gene. The distributions of the controls’ SMN1 copy numbers were 1 (3.1%), 2 (95.4%), and 3 (1.5%). Estimated copies of SMN2 were either 0 copies, 1 copies, 2 copies, 3 copies, or 4 copies. The number of patients with various SMA types (I and II) and controls are displayed in table (13). A significant difference was observed among the three clinical groups (SMA type I, II, and control) based on the findings; p value < 0.001. In addition, 83% of controls had 2 copies of SMN2 gene. In type I-SMA patients, 93% of patients had 1 copy or 2 copies of SMN2 gene. In type II SMA, 60 % of patients had 3 copies SMN2 gene. No patients with 0
Copy of SMN2 were found in their population. Only 3.1% of the control had no copies of the SMN2 gene. Only 46.7% of patients who had one copy of the SMN2 gene had type I-SMA. Only 7% of patients who had four copies of the SMN2 gene had type II-SMA. , We counted the copies of SMN1 and SMN2 in the three groups under investigation as well as in SMA patients and controls. A higher number of copies of SMN1 in an individual is linked to a lower number of copies of SMN2 in the overall population (controls); that is, in individuals who possess three copies of SMN1, the number of SMN2 copies reduced to zero in 3.1% of cases, whereas 3.1% individuals with one copy of SMN1 had four copies of SMN2 (p<0.001). SMN2 copy number was increased to three or four in a subset of SMN1 deletion/conversion controls, and in most SMA patients with a milder phenotype.
Combining the genotypes of SMN1 exons 7 & 8 and NAIP genes, Five genotypes were determined (Table 12), SMN1 exons 7 & 8 with NAIP del + 1 or 2 copies of SMN2 genotype were the most common type I patients, accounting for 53.3% (16/30) of patients. SMN1 exons 7 & 8 del with NAIP no del +2 or 3 or 4 copies of SMN2 genotype were the most common type II patients, accounting for 65.7% (23/35) of patients. SMN1 exons 7 & 8 with NAIP no del + 2 copy SMN2 genotype were the most common controls.
We sequenced the SMN1 gene for this work, SMN2 and NAIP genes on 10 of SMA patients (I&II) and 5 of controls in order to find mutations of SMA.
Table lists the SNPs having the highest significance (11). A total of 4 SNPs in SMN1, namely C -859G p.(Ala2Gly) was found in SMA patients as (Type 1 equals two copies of SMN2, while Type 2 equals three copies of SMN2.). Causes an amino acid change from Ala to Gly at position 2. this variant has previously been described as disease causing for Spinal muscular atrophy (Tan et al., 2020), G-711A was found to be most significant with identification in SMA type II = 3 copies of SMN2, T-1040A was found to be most significant with identification in SMA type I = 2 copies of SMN2, and T-1058A was found in SMA patients as (Type 1 is two copies of SMN2, and Type 2 is three copies of SMN2) (NG_008691.1).
Four variants that are also usually in SMN2, G-1102C, G-1119C, G-1093T and G-1094T were found to be most significant with identification in SMA type I = 2 copies (NM_022875.3) .
A total of 3 SNPs in NAIP, namely G-711A was found to be most significant with identification in SMA type II = 3 copies of SMN2, T-1040A was found to be most significant with identification in SMA type I = 1 copy of SMN2 with NAIP 5 deletion and T-1058A was found in SMA patients as (Type 1 equals two copies of SMN2, while Type 2 equals three copies of SMN2). (NM_001346870.2)
In summary, we conducted an investigation comparing the gene copy numbers and genetic polymorphisms of SMN1, SMN2, and NAIP genes in both SMA patients and healthy individuals in Egypt. Our findings demonstrate a strong correlation between SMN2 copy number and SMA disease severity, suggesting that the identification of SMN2 copy number could be an effective predictor of the type of SMA disease. Moreover, we discovered that the deletion of the NAIP gene was linked to SMA severity. Combining the analysis of NAIP deletion with the evaluation of SMN2 copy number enhances the predictive capacity of this approach for SMA severity. Alleles carrying a homozygous absence of SMN1 exons 7 and 8 in combination with NAIP exon 5 deletion are recognized as severe alleles. The gene structures of SMN and NAIP were also different between the SMA patients and healthy controls, exist and can affect the SMA phenotype.