Introduction

Neuromuscular diseases (NMD) are a category of neurological diseases that present a myriad of clinical manifestations affecting different components of the neuromuscular system, which is involved in the voluntary control of body movements.

NMDs are characterized by dysfunction or damage to anterior horn cells, muscles, neuromuscular junctions, or peripheral nerves1,2,3. Recently, with the advancement of genetic and metabolic testing, accompanied by the development of commercial (Next Generation Sequencing) gene panels specific for each phenotype, reaching a conclusive diagnosis has become easier than previously performed testing4,5,6,7,8,9,10,11,12. NGS panel have become a valuable component, along with clinical examination and neurophysiologic testing, in the diagnostic work up of patients with neuromuscular diseases4,5,6,11.

From a clinician’s standpoint, choosing a test for a patient can be challenging. Cost-effectiveness in tiered testing remains an issue, and the yield of each test varies depending on numerous factors such as disease phenotype, patient population and commercial lab capabilities7,8. Whole exome sequencing (WES) has been developed to cover a large number of genes and copy number variants (CNVs), with limited depth of coverage and bioinformatics approaches. While NGS gene panels are ideal for a single clear phenotype, they have limitations due to the variability of diseases and the number of genes selected for diagnosis7,8.

The effective identification of a gene mutation relies heavily on the selection of potential markers in the gene panel. Chromosomal microarray is another method used to evaluate such cases, with results varying based on the phenotype9,10. Additionally, Biochemical testing for inborn errors of metabolism is commonly used in the pediatric population as a first-tier screening test for many conditions, but its yield in NMDs is uncertain4,11,13. In the current era of personalized medicine, it is crucial for a clinician to promptly and cost-effectively diagnose patients. In the field of neuromuscular genetics, there is limited literature on the diagnostic utility of testing7.

Consanguineous marriages, or unions between closely related individuals, are common in Saudi Arabia. The high frequency is primarily driven by cultural, social, and economic factors, such as familial cohesion and economic benefits with rates ranging from 40 to 60% depending on the region and population studied14. Such marriages can strengthen family ties, but also increase the risk of autosomal recessive genetic disorders due to the inheritance of shared deleterious alleles from common ancestors15,16.

Research on neurological disorders, including epilepsy, intellectual disabilities, and certain neurodevelopmental conditions, has shown significantly higher prevalence where the rate of consanguinity is higher17,18,19,20,21,22,23,41.

In this study, we aimed to investigate the effectiveness of genetic and biochemical testing in the context of neuromuscular diseases with varying presentations, particularly within a highly consanguineous population.

Material and methods

After obtaining approval from the institutional review board, we conducted a retrospective review of the medical records of pediatric patients seen at two subspecialized pediatric neuromuscular disorders clinics at King Fahad Specialist Hospital in Dammam, Saudi Arabia, between 2013 and 2022. We specifically focused on patients under 16 years of age who presented with primary neuromuscular symptoms such as hypotonia, difficulty walking, delayed motor milestones, high creatine kinase levels, and exercise intolerance. Patients with incomplete data, those who only underwent serologic testing and individuals who did not receive follow-up care were excluded from the study. All data were meticulously entered into a Microsoft Excel spreadsheet by A. A. and reviewed by F.G., M. M and F.A.

Diagnosis of neuromuscular patients

The diagnosis of neuromuscular patients was based on a thorough review of patient history, physical examinations, and electrophysiological findings. Genetic testing was recommended by the attending pediatric neuromuscular neurologist and whole blood samples were collected for analysis. Patients already diagnosed with Duchenne Muscular Dystrophy (DMD), Spinal Muscular Atrophy (SMA), or Facioscapulohumeral muscular dystrophy (FSHD) through targeted gene testing were excluded from our study. Additionally, patients with autoimmune myasthenia gravis, which is diagnosed through serologic testing, and with syndromic hypotonia diagnosed prior to their visit to the neuromuscular disorder clinic were also excluded.

The determination of the pathogenicity of the variants was done through genetic testing. Mutational analysis was done through the reads of selected gene panels and whole exome sequencing approaches on the peripheral blood sample from the patients.

Metabolic and genetic testing

The following data were collected to determine the effectiveness of testing in our cohort: diagnosis, consanguinity, creatine kinase levels, metabolic and genetic test results and whether they are conclusive or not. Serum metabolic testing included plasma testing of ammonia, lactate, pyruvate, acylcarnitine profile, and amino acid levels, at the discretion of the treating physician.

Urine tests included an acylcarnitine profile and amino/organic acids as deemed necessary by the treating physician. Metabolic testing was conducted biochemically at our institution. Genetic testing included tests such as chromosomal micro-array analysis (CMA) performed by CGC Genetics using the Cytoscan HD platform from Affymetrix. Hybridization results were analyzed using ChAS Software (Chromosome Suite Analysis, Affymetrix). We utilized gene panels based on presentation, whole exome sequencing, and whole genome sequencing. Some patients underwent a panel followed by whole exome sequencing (WES) in a step-wise manner, while two patients underwent WES followed by panels as recommended by their attending neurologists. Genetic tests were carried out at CGC Genetics (CGC Genetics Inc., New Jersey, U.S.A.) or Centogene (Centogene, Rostock, Germany).

Both treating pediatric neurologists and clinical geneticists reviewed all variants of uncertain significance and likely pathogenic variants in the context of the clinical phenotype. Variants were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines24.

Results

After applying the inclusion and exclusion criteria, a retrospective review of the data for 130 patients referred to the neuromuscular clinics was conducted. Of these, 36 patients were excluded due to incomplete data (n = 2), targeted gene sequencing (n = 25), serologic testing only (n = 7), or having syndromic hypotonia diagnosed elsewhere (n = 2). Ultimately, the data for the remaining 94 patients were included and analyzed as depicted in Fig. 1 flowchart.

Fig. 1
figure 1

Flowchart.

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Phenotypic Characterization revealed that, sixteen patients presented with metabolic myopathy and rhabdomyolysis (17%), fourteen with hereditary spastic paraplegia (14.8%), thirteen with hypotonia (13.8%), thirteen with congenital myopathy (13.8%), twelve with peripheral neuropathy (12.7%), eleven patients with congenital muscular dystrophy (11.7%), seven patients presented with ataxia (7.4%), four patients with congenital myasthenia (4.2%), two patients presented with a phenotype suggestive of a channelopathy (2.1%), and another two with limb-girdle muscular dystrophy (2.1%) (Table 1).

Table 1 Phenotype distribution of individuals with identified pathogenic genes in our cohort.
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In our cohort, 78 patients had a positive family history of consanguinity (83%). 62 Patients underwent genetic panels (66%), 29 of them revealed a conclusive diagnosis (45%). Whole Exome Sequencing was done in 53 patients (56%) and was conclusive in 30 patients (56%).

Twenty-two Patients underwent both a gene panel and WES (23%) in a tiered manner; 9 (40%) of which yielded conclusive results with two had a post-WES gene panel revealed the diagnosis Thirty-one patients underwent CGH array (32%), only two of which showed abnormal results (2.1%) and nine patients had extensive regions of homozygosity, implying the high degree of consanguinity in our patient population (9.6%).

We had 37 patients who underwent metabolic testing (40%), none of them showed any conclusive results that aided in diagnosis. Overall, 58 patients (61%) had conclusive results after all testing, and in the remaining 36 patients, 20 of them showed variants of unknown significance (VOUS) (21%).

Within our patients who yielded conclusive results, we had 8 patients diagnosed with various subtypes of CMT, three patients with metabolic myopathies, and two with Emery-Dreifuss Muscular Dystrophy. All the others were diagnosed with various neuromuscular diseases (see Table 1 for details). The clinical characteristics of our 18 patients who had VOUS are described in Table 2.

Table 2 Clinical and genetic characteristics of patients with variants of unknown significance (VOUS) in the cohort.
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Discussion

Genetic testing is an important tool in the diagnostic journey of a patient with neuromuscular disease, typically starting with single gene testing if a clear diagnosis is suspected. With advances in NGS technology, commercial gene panels and WES have become the first-tier tests25,26,27,28. In our cohort, 61% of NMD patients had conclusive results and reached a definitive diagnosis.

In the clinical and practical setting, after phenotyping the patient, a clinician may choose between a commercial NGS gene panel or WES. Winder et al. tested 25,000 patients with NMDs using both targeted genes and gene panels, with a yield ranging between 4 and 33% based on the panel29. Our study showed an even higher rate of conclusive commercial panels (45%), which could potentially be due to the selection of specific gene panels in both studies or the complexity of the patient population in our tertiary-care facility.

WES has proven to be an important diagnostic tool for neuromuscular disorders, which pose a diagnostic challenge30. This increases the possibility of reaching a conclusive genetic diagnosis by identifying genes that were not previously associated with a specific NMD when other technologies have failed. It can also help exploring new genes. Furthermore, this strategy is useful for confirming a dual diagnosis in cases of complex congenital anomalies and undiagnosed cases31. Additionally, it is a cost-effective diagnostic tool that should be prioritized in the early diagnostic path of children with progressive neurological disorders32.

Previous studies have shown that WES has a yield of 49–63% in NMD patients33,34, and 52% in myopathy patients35. Our findings were similar, with 56% positive results, which is higher than gene panels. This indicates that WES can be very valuable in diagnosing patients presenting with an NMD condition and may even be used as an initial step before invasive electrophysiological testing, which can be challenging in pediatric patients.

Diagnosis of NMD is often challenging, particularly when it comes to genetic testing7. A common issue that arises during this process is the discovery of variants of unknown significance (VOUS), which was also reflected in our data (Table 2). We found that 19 patients had VOUS in commonly faced genes such as TTN, RYR1, and COL6A2. Unfortunately, this precludes patients from receiving a definitive diagnosis and requires further workup, such as segregation analysis for the parents as well as functional testing for the select variants to determine pathogenicity.

In the context of NMDs, Djordjevic et al. reported that metabolic testing can be used to screen for inborn errors of metabolism (IEM) that present with hypotonia36. It can also help diagnose metabolic myopathies, particularly fatty acid oxidation disorders, as noted by Tarnopolsky37. In some cases, it can distinguish between different types of mitochondrial diseases by the detection of various metabolites, as reported by Thompson et al.38. However, in our cohort, metabolic screening did not prove beneficial, which could be attributed to the absence of mitochondrial disorders in our patients and the fact that metabolic myopathy patients were not in crises during at the time of testing.

In contrast to a previously reported study by Piluso et al.9, the utility of CGH array was limited in establishing a diagnosis for our cohort patients. Nine out of thirty-one patients exhibited regions of excessive homozygosity, indicating a high consanguinity rate. High rates of intermarriage among relatives causing a genetic signature of consanguinity, have been strongly linked to a higher prevalence of neurological disorders in populations with excessive homozygosity15,16. Inheriting identical alleles from common ancestors can lead to an elevated risk of autosomal recessive disorders, which are particularly common in neurological diseases such as epilepsy, intellectual disabilities, and neurodegenerative conditions15,18,39,40,41. Studies have reported that consanguineous unions is associated with a greater incidence of pathogenic conditions due to this excessive homozygosity42,43,44. Homozygous deleterious alleles can contribute to both common and rare neurological disorders within consanguineous populations. This genetic phenomenon poses a significant public health concern, as the accumulation of these alleles can lead to higher prevalence of neurological disorders45,46,47.

Limitations

This study was limited by the small number of recruited patients, which mainly caused difficulties in recruiting patients and obtaining data through Electronic Medical Records (EMR). Future studies can be conducted on a multicenter level to increase the number of patients in our population.

Conclusion

In conclusion, our study described the diagnostic yield of different types of genetic testing for NMD patients, in a highly consanguineous Saudi population. The yield was higher in WES than in NGS panels, with CGH array and metabolic testing showing no yield at all. These results, although limited by sample size, could favor the use of WES as a first-tier test in the work up of a patient presenting with a neuromuscular disease.