The genetic basis of early-onset hereditary ataxia in Iran: results of a national registry of a heterogeneous population.

BACKGROUND: To investigate the genetics of early-onset progressive cerebellar ataxia in Iran, we conducted a study at the Children's Medical Center (CMC), the primary referral center for pediatric disorders in the country, over a three-year period from 2019 to 2022. In this report, we provide the initial findings from the national registry. METHODS: We selected all early-onset patients with an autosomal recessive mode of inheritance to assess their phenotype, paraclinical tests, and genotypes. The clinical data encompassed clinical features, the Scale for the Assessment and Rating of Ataxia (SARA) scores, Magnetic Resonance Imaging (MRI) results, Electrodiagnostic exams (EDX), and biomarker features. Our genetic investigations included single-gene testing, Whole Exome Sequencing (WES), and Whole Genome Sequencing (WGS). RESULTS: Our study enrolled 162 patients from various geographic regions of our country. Among our subpopulations, we identified known and novel pathogenic variants in 42 genes in 97 families. The overall genetic diagnostic rate was 59.9%. Notably, we observed PLA2G6, ATM, SACS, and SCA variants in 19, 14, 12, and 10 families, respectively. Remarkably, more than 59% of the cases were attributed to pathogenic variants in these genes. CONCLUSIONS: Iran, being at the crossroad of the Middle East, exhibits a highly diverse genetic etiology for autosomal recessive hereditary ataxia. In light of this heterogeneity, the development of preventive strategies and targeted molecular therapeutics becomes crucial. A national guideline for the diagnosis and management of patients with these conditions could significantly aid in advancing healthcare approaches and improving patient outcomes.

A comparative study on prophylactic efficacy of cinnarizine and amitriptyline in childhood migraine: a randomized double-blind clinical trial.

BACKGROUND: Pediatric migraine prophylaxis is indicated when headaches are frequent and/or disabling. We aimed to conduct a study to compare the efficacy of cinnarizine and amitriptyline in pediatric migraine prophylaxis. METHODS: In a randomized, double-blind trial, patients aged 4-17 years with migraine who were eligible for prophylaxis enrolled. The primary outcome was a reduction response rate of ≥50% with p < 0.005 with respect to headache characteristics. The secondary outcome was migraine disability assessment. We evaluated patients every four weeks for three months: T1: week 4, T2: week 8 and T3: week 12. The safety profile was also assessed. RESULTS: Thirty patients were randomly assigned to each group. However, 43 patients completed the trial. Headache frequency decreased in amitriptyline group more effectively in T1 (p = 0.004). Amitriptyline was more successful in reducing the headache duration in all three periods (p < 0.005). There was no significant difference in severity improvement and reducing disability score between the two groups (p > 0.005). No serious adverse events were observed. CONCLUSIONS: Both medications are effective in ameliorating migraine headaches and related disabilities. However, amitriptyline appears be a preferable option over cinnarizine, given its faster onset of action, efficacy in reducing headache duration and longer-lasting effects.Trial Registration: The study was registered with the Iranian Registry of Clinical Trials (IRCT) under the code IRCT-20191112045413N1.

High genetic heterogeneity of leukodystrophies in Iranian children: the first report of Iranian Leukodystrophy Registry.

Leukodystrophies (LDs) are a heterogeneous group of progressive neurological disorders and characterized by primary involvement of white matter of the central nervous system (CNS). This is the first report of the Iranian LD Registry database to describe the clinical, radiological, and genomic data of Persian patients with leukodystrophies. From 2016 to 2019, patients suspicious of LDs were examined followed by a brain magnetic resonance imaging (MRI). A single gene testing or whole-exome sequencing (WES) was used depending on the neuroradiologic phenotypes. In a few cases, the diagnosis was made by metabolic studies. Based on the MRI pattern, diagnosed patients were divided into cohorts A (hypomyelinating LDs) versus cohort B (Other LDs). The most recent LD classification was utilized for classification of diagnosed patients. For novel variants, in silico analyses were performed to verify their pathogenicity. Out of 680 registered patients, 342 completed the diagnostic evaluations. In total, 245 patients met a diagnosis which in turn 24.5% were categorized in cohort A and the remaining in cohort B. Genetic tests revealed causal variants in 228 patients consisting of 213 variants in 110 genes with 78 novel variants. WES and single gene testing identified a causal variant in 65.5% and 34.5% cases, respectively. The total diagnostic rate of WES was 60.7%. Lysosomal disorders (27.3%; GM2-gangliosidosis-9.8%, MLD-6.1%, KD-4.5%), amino and organic acid disorders (17.15%; Canavan disease-4.5%, L-2-HGA-3.6%), mitochondrial leukodystrophies (12.6%), ion and water homeostasis disorders (7.3%; MLC-4.5%), peroxisomal disorders (6.5%; X-ALD-3.6%), and myelin protein disorders (3.6%; PMLD-3.6%) were the most commonly diagnosed disorders. Thirty-seven percent of cases had a pathogenic variant in nine genes (ARSA, HEXA, ASPA, MLC1, GALC, GJC2, ABCD1, L2HGDH, GCDH). This study highlights the most common types as well as the genetic heterogeneity of LDs in Iranian children.

Clinical and Molecular Findings of Autosomal Recessive Spastic Ataxia of Charlevoix Saguenay: an Iranian Case Series Expanding the Genetic and Neuroimaging Spectra.

Autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is now increasingly identified from all countries over the world, possibly rendering it one of the most common autosomal recessive ataxias. Here, we selected patients harboring SACS variants, the causative gene for ARSACS, in a large cohort of 137 patients with early-onset ataxia recruited from May 2019 to May 2021 and were referred to the ataxia clinic. Genetic studies were performed for 111 out of 137 patients (81%) which led to a diagnostic rate of 72.9% (81 out of 111 cases). Ten patients with the molecular diagnosis of ARSACS were identified. We investigated the phenotypic and imaging spectra of all confirmed patients with ARSACS. We also estimated the frequency of ARSACS in this cohort and described their clinical and genetic findings including seven novel variants as well as novel neuroimaging findings. While the classic clinical triad of ARSACS is progressive cerebellar ataxia, spasticity, and sensorimotor polyneuropathy, it is not a constant feature in all patients. Sensorimotor axonal-demyelinating neuropathy was detected in all of our patients, but spasticity and extensor plantar reflex were absent in 50% (5/10). In all patients, brain magnetic resonance imaging (MRI) showed symmetric linear hypointensities in the pons (pontine stripes) and anterior superior cerebellar atrophy as well as a hyperintense rim around the thalami (thalamic rim). Although infratentorial arachnoid cyst has been reported in ARSACS earlier, we report anterior temporal arachnoid cyst in two patients for the first time, indicating that arachnoid cyst may be an associated imaging feature of ARSACS. We also extended molecular spectrum of ARSACS by presenting 8 pathogenic and one variant of unknown significance (VUS) sequence variants, which 7 of them have not been reported previously. MetaDome server confirmed that the identified VUS variant was in the intolerant regions of sacsin protein encoded by SACS.

Efficacy and safety of miglustat in the treatment of GM2 gangliosidosis: A systematic review.

BACKGROUND: Since the results of previous studies regarding the safety and efficacy of miglustat in GM2 gangliosidosis (GM2g) were inconsistent, we aimed to assess miglustat therapy in GM2g patients. METHODS: This study followed the latest version of PRISMA. We included the observational or interventional studies reporting GM2g patients under miglustat therapy by searching PubMed, Web of Science, and Scopus. Data extracted included the natural history of individual patient data, as well as the safety and efficacy of miglustat in GM2g patients. The quality assessment was performed using the Joanna Briggs Institute Critical Appraisal checklist. RESULTS: A total of 1023 records were identified and reduced to 621 after removing duplicates. After screening and applying the eligibility criteria, 10 articles and 2 abstracts met the inclusion criteria. Overall, the studies represented 54 patients with GM2g under treatment with miglustat and 22 patients with GM2g in the control group. Among patients with available data, 14 and 54 have been diagnosed with Sandhoff disease and Tay-Sachs disease, respectively. Patients included in this review consisted of 23 infantile, 4 late-infantile, 18 juvenile, and 31 adult-onset GM2g. CONCLUSIONS: Although miglustat should not be considered a definite treatment for GM2g, it appears that patients, particularly those with infantile or late-infantile GM2g, could benefit from miglustat therapy to some extent. We also make some suggestions regarding future studies presenting their findings in a standard format to facilitate pooling the available data in such rare diseases for a more comprehensive conclusion.

Expanding phenotype heterogeneity of NARS2 by presenting subdural hematoma and parenchymal hemorrhage.

BACKGROUND: NARS2 encodes mitochondrial Asparaginyl-tRNA Synthetase 2, which catalyzes the aminoacylation of tRNA-Asn in the mitochondria. To date, 24 variants have been reported in NARS2 gene in 35 patients. The phenotypic variability of NARS2-associated disorder is broad, ranging from neurodevelopmental disorders to hearing loss. In this study, we report some novel imaging findings in an Iranian patient suffering from epileptic encephalopathy, caused by a previously reported variant, c.500A > G; p.(His167Arg), in NARS2. METHODS: The spectrum of clinical manifestations of two Iranian patients was investigated and genetic analysis was performed by Whole-exome sequencing (WES). Additionally, we also reviewed the literature and summarized the phenotypes of previously reported patients with variants in the NARS2 gene. RESULTS: Here, we present the phenotypic and genetic features of 2 unrelated Iranian infants presented with neurodevelopmental delay, seizures, hearing impairment, feeding problems, elevated serum lactate levels in addition to subdural hematoma and cerebral parenchymal hemorrhage in the brain magnetic resonance imaging (MRI) of one of the patients. Genetic analysis revealed a biallelic missense variant in NARS2: c.500A > G; p.(His167Arg). We described the subdural hematoma and cerebral parenchymal hemorrhage of the brain for the first time. CONCLUSIONS: Our study provides new clinical findings, subdural hematoma, and parenchymal hemorrhage, in NARS2-related disorders. Our findings along with previous studies provide more evidence of the clinical presentation of the disease caused by pathogenic variants in NARS2. Expanding the clinical spectrum increases the diagnostic rate of molecular testing and improves the quality of counseling for at-risk couples.

Characteristics of disease progression and genetic correlation in ambulatory Iranian boys with Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy in the pediatric population. The manifestations of this disease include progressive muscle weakness, gait dysfunction, and motor impairment, leading to a loss of ambulation by the age of 13 years. Molecular diagnosis is the standard diagnostic tool for DMD. This study aimed to investigate disease progression and genetic patterns in Iranian ambulant boys and to find the correlation between genotypes and motor function phenotypes. METHODS: This study was performed on 152 DMD patients. Clinical history, including the disease phenotype, steroid therapy, and the North Star Ambulatory Assessment (NSAA) score, was taken for all the patients. Molecular diagnoses were confirmed by multiplex ligation-dependent probe amplification and next-generation sequencing tests. RESULTS: A total of 152 Iranian DMD patients were examined in this study. The mean age at the time of disease onset was 4.04 ± 2.00 years, and the mean age at diagnosis was 5.05 ± 2.08 years. The mean age of ambulation loss was 10.9 years. Contracture was reported in 38.9% of cases. In terms of age, the mean total NSAA score showed a peak at 4 years of age, with a mean NSAA score of 24. Annual changes in the NSAA score were determined for all cases, based on the mutation type and exon site. Deletion mutation was found in 79.1% of cases, duplication in 6.8%, nonsense in 12.8%, and splice site in 1.4%. The most common single exon deletion was exon 44 (5.3%), and the most common multiexon deletions were attributed to exons 45-50 and exons 45-52 (4.6%). The results did not indicate any correlation between the mutation type and age at the time of disease onset, loss of ambulation age, and wheelchair dependence; however, a significant association was found between contracture and mutation type. The results showed a significant difference in the NSAA score between the deletion and nonsense groups at the age of 3 years (P = 0.04). No significant correlation was found between the phenotype and exon site. Overall, 91.1% of the study population had a history of corticosteroid use, and 54.1% showed compliance with rehabilitation therapy. CONCLUSION: This study demonstrated the phenotypes and mutational features of Iranian DMD boys and provided information regarding the natural motor history of the disease, disease progression, diagnosis, and status of DMD management in Iran. The present findings can promote the development of clinical trials and future advanced molecular therapies in Iran.

The quality of life in children with spinal muscular atrophy: a case-control study.

OBJECTIVES: This study aimed to analyze the health-related quality of life (HRQoL) of patients with spinal muscular atrophy (SMA) based on the type of SMA, demographic and clinical features and compare HRQoL of these patients with a matched healthy control group.  METHODS: This was a case-control study of Patients with SMA in Iran. Sixty-six patients with SMA type II and III aged 8-18 years and also 264 healthy age, sex, and socio-economic matched individuals were enrolled. To assess the quality of life, we used the Persian version of the KIDSCREEN-27. RESULTS: The health-related quality of life between children with type II and type III SMA was not significant in all 5 subscales. However, HRQoL in healthy children was significantly higher than in SMA children in all 5 subscales. CONCLUSION: The quality of life in children with SMA was lower than the healthy control group in all subscales, and physical well-being and psychosocial aspects are the main domains of life impaired by SMA disease. However, no significant difference between the quality of life in children with SMA type II and type III was observed.

Homozygous in-frame variant of SCL6A3 causes dopamine transporter deficiency syndrome in a consanguineous family.

The human dopamine transporter (hDAT) participates in dopamine homeostasis by clearing dopamine from the extracellular space using secondary active transport. Dysregulation of hDAT has been reported to be associated with different neuropsychiatric disorders. Dopamine transporter deficiency syndrome (DTDS) is a complex disease caused by defects in dopamine uptake within the synaptic cleft and patients manifest parkinsonian features. The extracellular loops are crucial for DAT activity and defects in these regions disturb dopamine transport. In the present study, a 3.5-year-old female in a consanguineous Iranian family with an initial diagnosis of gait imbalance and speech delay has been identified. We utilized whole-exome sequencing (WES) to identify the possible genetic defect(s). WES identified a novel homozygous in-frame indel variant, c.1139_1150del; p.(Gly380_Lys384delinsGlu), in the SLC6A3 gene (NM_001044.4), as the most likely disease-susceptibility variant. This variant is located in extracellular loop 4 (EL4) of the DAT protein. Our study highlights the role of extracellular loops and shows the EL4 of hDAT as a critical region for the protein activity. The identified variant in the EL4 region of DAT is predicted to compromise DAT function and may lead to DTDS in this case. However, complementary studies are required to confirm.

Hypomyelinating Leukodystrophy with Spinal Cord Involvement Caused by a Novel Variant in RARS: Report of Two Unrelated Patients.

Leukodystrophies are heterogeneous group of genetic white matter disorders with a wide range of neurologic and systemic manifestations. Defects in genes encoding aminoacyl tRNA (transfer ribonucleic acid) synthetase enzymes (aaRSs) are recently identified as the etiology of some leukodystrophies. Herein, we described two unrelated children referred to Children's Medical Center, Tehran, Iran, with developmental delay, nystagmus, seizures, psuedo-bulbar palsy and dystonia. Whole exome sequencing (WES) in both patients identified a homozygous (c.2T > C) variant in exon one of RARS gene, encoding cytoplasmic arginyl-tRNA synthetase. Our finding was confirmed by segregation analysis. In silico analyses of the c.2T > C variant showed its possible pathogenic role due to the absence of the start codon. Severe hypomyelination was the common neuroimaging finding of both cases. Spinal cord involvement was found in one of our patients which was not previously reported in studies. We, therefore, showed that RARS-related hypomyelination might affect spinal cord.

An overview of early-onset cerebellar ataxia: a practical guideline.

Early onset ataxias (EOAs) are a heterogeneous group of rare neurological disorders that not only involve the central and peripheral nervous system but also involve other organs. They are mainly manifested by degeneration or abnormal development of the cerebellum occurring before the age of 25 years and typically the pattern of inheritance is autosomal recessive.The diagnosis of autosomal recessive cerebellar ataxias (ARCAs) is confirmed by the clinical, laboratory, electrophysiological examination, neuroimaging findings, and mutation analysis when the causative gene is detected. Correct diagnosis is crucial for appropriate genetic counseling, estimating the prognosis, and, in some cases, pharmacological intervention. The wide variety of genotypes with a heterogeneous phenotypic manifestation makes the diagnostic work-up challenging, time-consuming, and expensive, not only for the clinician but also for the children and their parents. In this review, we focused on the step-by-step approach in which cerebellar ataxia is a prominent sign. We also outline the most common disorders in ataxias with early-onset manifestations.

TPP1 Variants in Iranian patients: A Novel Pathogenic Homozygous Variant Causing Neuronal Ceroid Lipofuscinosis 2.

Introduction: TPP1 variants have been identified as a causative agent of neuronal ceroid lipofuscinosis 2 disease, that ataxia is one of its clinical features. Therefore, here, molecular study of TPP1 variants is presented in an Iranian cohort and a novel pathogenic variant is described. Methods: This investigation was conducted as a cross-sectional study in a tertiary referral hospital, Children's Medical Center, Pediatrics Center of Excellence. Clinical presentations and pedigrees were documented. Patients with cerebellar ataxia were enrolled in this study. Next-generation sequencing was applied to confirm the diagnosis. Segregation and bioinformatics analyses were also done for the variants using Sanger sequencing. Results: Forty-five patients were included in our study. The mean age of onset was 104 (+55.60) months (minimum = 31 months, maximum = 216 months). The majority of cases (73.3%) were born to consanguineous parents and only 1 patient (2.2%) had an affected sibling. Of the 45 patients, only 1 patient with a novel pathogenic variant (c.1425_1425+1delinsAT, p.A476Cfs*15) in the TPP1 gene was identified. Discussion: The main strength of current study is the relatively large sample size. Besides, a novel pathogenic variant could be important toward the diagnosis and management of this condition. With significant advances in various therapies, early diagnosis could improve the treatments using personalized-based medicine.

A comprehensive study of mutation and phenotypic heterogeneity of childhood mitochondrial leukodystrophies.

OBJECTIVE: Mitochondrial leukodystrophies (MLs) are mainly caused by impairments of the mitochondrial respiratory chains. This study reports the mutation and phenotypic spectrum of a cohort of 41 pediatric patients from 39 distinct families with MLs among 320 patients with a molecular diagnosis of leukodystrophies. METHODS: This study summarizes the clinical, imaging, and molecular data of these patients for five years. RESULTS: The three most common symptoms were neurologic regression (58.5%), pyramidal signs (58.5%), and extrapyramidal signs (43.9%). Because nuclear DNA mutations are responsible for a high percentage of pediatric MLs, whole exome sequencing was performed on all patients. In total, 39 homozygous variants were detected. Additionally, two previously reported mtDNA variants were identified with different levels of heteroplasmy in two patients. Among 41 mutant alleles, 33 (80.4%) were missense, 4 (9.8%) were frameshift (including 3 deletions and one duplication), and 4 (9.8%) were splicing mutations. Oxidative phosphorylation in 27 cases (65.8%) and mtDNA maintenance pathways in 8 patients (19.5%) were the most commonly affected mitochondrial pathways. In total, 5 novel variants in PDSS1, NDUFB9, FXBL4, SURF1, and NDUSF1 were also detected. In silico analyses showed how each novel variant may contribute to ML pathogenesis. CONCLUSIONS: The findings of this study suggest whole-exome sequencing as a strong diagnostic genetic tool to identify the causative variants in pediatric MLs. In comparison between oxidative phosphorylation (OXPHOS) and mtDNA maintenance groups, brain stem and periaqueductal gray matter (PAGM) involvement were more commonly seen in OXPHOS group (P value of 0.002 and 0.009, respectively), and thinning of corpus callosum was observed more frequently in mtDNA maintenance group (P value of 0.042).

Phenotype and genotype heterogeneity of PLA2G6-associated neurodegeneration in a cohort of pediatric and adult patients.

BACKGROUND: Phospholipase-associated neurodegeneration (PLAN) caused by mutations in the PLA2G6 gene is a rare neurodegenerative disorder that presents with four sub-groups. Infantile neuroaxonal dystrophy (INAD) and PLA2G6-related dystonia-parkinsonism are the main two subtypes. In this cohort, we reviewed clinical, imaging, and genetic features of 25 adult and pediatric patients harboring variants in the PLA2G6. METHODS: An extensive review of the patients' data was carried out. Infantile Neuroaxonal Dystrophy Rating Scale (INAD-RS) was used for evaluating the severity and progression of INAD patients. Whole-exome sequencing was used to determine the disease's underlying etiology followed by co-segregation analysis using Sanger sequencing. In silico prediction analysis based on the ACMG recommendation was used to assess the pathogenicity of genetic variants. We aimed to survey a genotype-genotype correlation in PLA2G6 considering all reported disease-causing variants in addition to our patients using the HGMD database and the chi-square statistical approach. RESULTS: Eighteen cases of INAD and 7 cases of late-onset PLAN were enrolled. Among 18 patients with INAD, gross motor regression was the most common presenting symptom. Considering the INAD-RS total score, the mean rate of progression was 0.58 points per month of symptoms (Standard error 0.22, lower 95% - 1.10, and upper 95% - 0.15). Sixty percent of the maximum potential loss in the INAD-RS had occurred within 60 months of symptom onset in INAD patients. Among seven adult cases of PLAN, hypokinesia, tremor, ataxic gate, and cognitive impairment were the most frequent clinical features. Various brain imaging abnormalities were also observed in 26 imaging series of these patients with cerebellar atrophy being the most common finding in more than 50%. Twenty unique variants in 25 patients with PLAN were detected including nine novel variants. Altogether, 107 distinct disease-causing variants from 87 patient were analyzed to establish a genotype-phenotype correlation. The P value of the chi-square test did not indicate a significant relationship between age of disease onset and the distribution of reported variants on PLA2G6. CONCLUSION: PLAN presents with a wide spectrum of clinical symptoms from infancy to adulthood. PLAN should be considered in adult patients with parkinsonism or cognition decline. Based on the current knowledge, it is not possible to foresee the age of disease onset based on the identified genotype.

The First Report of Iranian Registry of Patients with Spinal Muscular Atrophy.

BACKGROUND: Insufficient amounts of survival motor neuron protein is leading to one of the most disabling neuromuscular diseases, spinal muscular atrophy (SMA). Before the current study, the detailed characteristics of Iranian patients with SMA had not been determined. OBJECTIVE: To describe the key demographic, clinical, and genetic characteristics of patients with SMA registered in the Iranian Registry of SMA (IRSMA). METHODS: IRSMA has been established since 2018, and the demographic, clinical, and genetic characteristics of patients with SMA were recorded according to the methods of treat neuromuscular disease (TREAT-NMD) project. RESULTS: By October 1, 2022, 781 patients with 5q SMA were registered. Of them, 164 patients died, the majority of them had SMA type 1 and died during the first 20 months of life. The median survival of patients with type 1 SMA was 23 months. The consanguinity rate in 617 alive patients was 52.4%, while merely 24.8% of them had a positive family history. The most common type of SMA in live patients was type 3. Morbidities were defined as having scoliosis (44.1%), wheelchair dependency (36.8%), tube feeding (8.1%), and requiring mechanical ventilation (9.9%). Most of the registered patients had a homozygous deletion of SMN1, while the frequency of patients with higher copy numbers of SMN2, was less in more severe types of the disease. Earlier onset of the disease was significantly seen in patients with lower copy numbers of SMN2. The neuronal apoptosis inhibitory protein (NAIP) gene deletion was associated with a higher incidence of more severe types of SMA, higher dependency on ventilators, tube feeding, and earlier onset of the disease. CONCLUSIONS: The IRSMA is the first established Iranian nationwide registry of patients with SMA. Using this registry, decision-makers, researchers, and practitioners can precisely understand the epidemiology, characteristics, and genetics of patients with SMA in Iran.

Follow-up of 25 patients with treatable ataxia: A comprehensive case series study.

Autosomal recessive cerebellar ataxias are a group of heterogeneous early-onset progressive disorders that some of them are treatable. We performed a 4-year follow-up for 25 patients who had treatable ataxia. According to our study, patients would benefit from early detection of treatable ataxia, close observation, and follow-up.

Genetic Analysis of Forty MLPA-Negative Duchenne Muscular Dystrophy Patients by Whole-Exome Sequencing.

This manuscript aimed to determine the underlying point mutations causing Duchenne muscular dystrophy (DMD) in a heterogeneous group of Iranian patients, who are clinically suspected. Whole-exome sequencing was utilized to detect disease-causing variants in 40 MLPA-negative DMD patients. Disease-causing variants were detected in the DMD gene in 36/40 of the patients (90%), and 4/40 of them (10%) remained undiagnosed. WES analysis revealed that nonsense variant was the most common type in our study (23/36 of the cases). Besides, 12/36 of the cases had frameshift variant, and one of the patients had a likely pathogenic splice variant in the DMD gene. Carrier testing revealed that 21/40 of the mothers had the identified variant. Therefore, most variants were inherited (58.3%), while 19/40 were de novo (41. 7%). The present study has demonstrated the importance of performing WES to detect disease-causing point mutations in MLPA-negative DMD patients and to identify carrier females. Due to regulatory challenges, the clinical development of therapeutic approaches is time-consuming and may not be available to all patients shortly. Therefore, it appears that the techniques used to accurately detect disease-causing variants in carrier mothers are a more efficient solution to prevent the increased prevalence of DMD.