Abstract
Introduction
Spinal Muscular Atrophy (SMA) is a genetic disorder caused by the loss of the survival motor neuron (SMN1) gene in over 95% of cases. Additionally, mutations in genes associated with the SMA chromosomal region can influence disease progression. Aim: To analyze the status of the NAIP and GTF2H2 genes in correlation with SMA.
Material and methods
The study included 105 patients suspected for SMA of which 50 with confirmed with SMA and 55 without causative deletions, and 107 healthy, unrelated individuals. The molecular genetics methods used were mPCR, PCR-RFLP and MLPA.
Results
From 105 patients, 50 were confirmed with SMA. In this group were identified in 8 patients (16%) with a homozygous deletion of exon 5 of the NAIP gene, 4 patients (8%) had a heterozygous status, and 2 (4%) had duplications. In the rest of the patients (55), in which deletions of SMN1 exon 7 were not identified, homozygous deletion of exon 5 of the NAIP gene was established in one patient (2%), 3 patients (5%) had duplications of exon 5 of the NAIP gene, and one patient had 5 copies of the NAIP gene. In the 107 healthy controls, one patient (1%) was identified with a deletion of exon 5 of the NAIP gene. None of the patients with combined deletions of SMN1 and NAIP had deletions in GTF2H2.
Conclusions
The frequency of deletions in the NAIP gene was found to be higher in the SMA patient group compared to the control group. Thus, a significant relationship was identified, the P value being <0.00001. The significance threshold was set at p<0.05. The genetic patterning of genes associated with SMA is an important aspect in the study of molecular pathophysiology and assessment of disease prognosis, especially in the approach to gene therapies.
Article highlights
What is not yet known on the issue addressed in the submitted manuscript
The worldwide understanding of Spinal Muscular Atrophy (SMA) genetics remains incomplete, with ongoing research needed to uncover the precise molecular mechanisms, global variations in genetic patterns, and the full spectrum of genotype-phenotype correlations. Additionally, the therapeutic implications of genetic profiles and the long-term outcomes of individuals with SMA, especially in complex cases, are areas that require further investigation to enhance SMA diagnosis and treatment strategies.
The research hypothesis
The research endeavors to explore the relationship between the genetic status of the NAIP and GTF2H2 genes, located in the SMA chromosomal region, and the diversity of mutations associated with Spinal Muscular Atrophy (SMA).
The novelty added by manuscript to the already published scientific literature
It has been demonstrated that there is a significant correlation between the presence of deletions in the NAIP gene and the occurrence of Spinal Muscular Atrophy (SMA), suggesting that NAIP gene deletions may play a crucial role in SMA susceptibility and severity. Also this research aims to reflect the deletion profile of the patients from Moldavian population in relation to the prevalence of deletions in the genes associated with Spinal Muscular Atrophy (SMA).
Full text
Introduction
Spinal muscular atrophy (SMA) is an autosomal recessive disorder that causes muscular atrophy and hypotonia [1, 2]. In over 95% of cases, SMA is caused by homozygous deletion of exon 7 in the SMN1 gene [3-5]. However, mutations in other genes in the SMA region can contribute to the disease, such as SMN2, NAIP, and GTF2H2 genes [6-8].
The NAIP gene (OMIM: 600355) [9] is located near the SMN gene and is also duplicated in the 5ql3 region. However, the copy associated with deletions in SMA patients can be distinguished because only this copy contains exon 5 [10-12]. Therefore, in some studies, deletion of exon 5 is present in approximately 50% of patients with SMA type I and ~20% in patients with type II and type III [13]. Experiments demonstrating that the NAIP gene is responsible for expressing a protein that suppresses cellular apoptosis support the idea that the protein acts as a negative regulator of motor neuron apoptosis [14]. When this protein is deficient or absent, it contributes to the SMA phenotype [15-17]. Thus, a moderate correlation has been demonstrated between mutations in the NAIP gene and the pathophysiology of SMA, especially when considered together with the number of SMN2 copies, particularly in SMA types I, II, and III [18].
The gene GTF2H2 (OMIM: 601748) a subunit of the basal transcription factor TFIIH, involved in the transcription process, DNA repair mechanisms and probably in other cellular processes was also characterized and located in the SMA region.
Thus, studies were reported on unrelated patients with SMA that showed that large deletions involving SMN, NAIP, GTF2H2 gene loci are associated with the most severe SMA phenotype (SMA type I) [20, 21]. The identification of deletions in these genes can be performed by different molecular genetics methods such as PCR, qPCR or MLPA [13, 17, 21].
Objective
The aim of this study was to analyze the profile of modifier genes associated with SMA, such as NAIP and GTF2H2, in patients with SMA.
Material and methods
The study was conducted at the Institute of Mother and Child, Human Molecular Genetics Laboratory. A total of 105 patients suspected of having SMA were enrolled in the study, including 50 patients with a molecular-genetic diagnosis of SMA, 55 patients with hypotonia but without confirmed molecular-genetic SMA, and 107 unrelated healthy individuals from the Human Molecular Genetics Laboratory's database. All patients have signed the consent for participation (approved by Research Ethics Committee of Nicolae Testemițanu State University of Medicine and Pharmacy, Act No. 3, from February 16, 2021). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex ligation-dependent probe amplification (MLPA) (The innovation Act “The method of diagnosing Spinal Muscular Atrophy by the MLPA technique” No. 483 from 17.03.2022) were used to detect deletions of exons 7 and 8 of the SMN1 gene for diagnostic purposes. Multiplex polymerase chain reaction (mPCR) was used for exon 5 of the NAIP gene and exon 4 of the GTF2H2 gene. Primers for identifying mutations in exon 5 of NAIP (The innovation Act „ Diagnostic method for the deletion of exon 5 of the NAIP gene based on the PCR genetic molecular technique” No.517 from 14.03.2023) and exon 4 of GTF2H2 through mPCR (The innovation Act "Diagnostic method of deletion of exon 4 of the GTF2H2 gene based on the PCR genetic molecular technique" No. 516 from 14.03.2023) were custom-designed, along with the parameterization of the amplification program. The innovative diagnostic methods used, as outlined in the acts mentioned above, contain the detailed protocol information, safeguarding that more specific data will not be made public to facilitate the filing of a patent application. Statistical analyzes were calculated via Social Science Statistics (Online) [22].
