Spinal Muscular Atrophy (SMA)

What is SMA disease?

SMA (Spinal muscular atrophy) is an inherited disorder characterized by the progressive and irreversible degeneration of motor neurons in the cells located in the anterior horn of the spinal cord and the brain stem nuclei. This disease displays clinical heterogeneity, exhibiting a variety of symptoms that can manifest at different times, ranging from prenatal stages to adulthood depending on the specific type of SMA. The primary defining feature of this condition is the weakening and gradual deterioration of organs. Clinical signs include Fasciculation, abnormal reflexes, scoliosis, stiffening of joints, as well as difficulties in respiration and feeding.

While SMA is considered to be a rare disease, it is actually one of the most prevalent genetic causes of infant mortality. Multiple genes are implicated in SMA, with the mutation in the SMN1 (Survival Motor Neuron 1) gene being particularly significant. Despite being categorized as a rare genetic disorder, statistics demonstrate an increasing number of individuals affected by SMA within the country. The financial burden of treating this condition is exorbitant, compounded by the scarcity of necessary medications. Consequently, a significant number of children die due to this genetic disorder each year.

SMA disease types and their symptoms

There are different classifications of spinal muscular atrophy disorders that occur due to mutations in the same genes and the symptomes of the disease occur at various stages of life. The classification of SMA disease is based on the time of onset and the severity of symptoms, which are divided into five categories:

SMA 0

This particular variant of SMA ailment is the most severe and the rarest form of disease which observed in newborns. It is worth mentioning that these affected infants frequently die due to acute respiratory difficulties within the initial months following birth, hence it is possible that the primary cause of their affliction may remain undiagnosed.

Symptoms:

Severe neonatal hypotonia
Areflexia
Atrial septal defects
Respiratory failure at the time of birth
Severe weakness
Decrease fetal movements
Arthrogryposis
Facial dipelgia or bilateral facial paralysis
Loss of respiratory muscle strength
Bell-shaped upper body
weak cries, weak coughs and accumulation of secretions in the lungs or throat in affected infants

SMA I

SMA type 1, also known as Werdnig-Hoffmann disease, is a prevalent form of this hereditary condition, accounting for approximately 80% of affected individuals. The onset of various symptoms occurs within the first few months after birth, ultimately resulting in premature mortality during early childhood.

Cause:

homozygous deletion of exon 7 or 8, or exon 7 of the SMN gene in 96% of SMA I cases
Homozygous deletion of NAIP gene (neuronal apoptosis inhibitory protein) in 46% of cases
Presence of 1 copy of SMN2 (Survival Motor Neuron2) gene in 96% of cases

Symptoms:

Difficulty in swallowing and sucking
Mild joint spasms
loss of head control
severe muscle weakness
Weak muscle tone
Loss of respiratory muscle strength
weak cries, weak coughs and accumulation of secretions in the lungs or throat in affected infants

SMA II

Spinal Muscular Atrophy type 2, also known as Debowitz disease, frequently appears in infants aged 6 to 12 months after birth. During this time, affected children require assistance in performing tasks such as sitting, standing, and walking. The symptoms associated with this particular form of SMA are of moderate severity, and the lifespan of individuals affected by it varies. Approximately 70% of patients can survive until the age of 25.

Cause:

Homozygous deletion of exon 7 or 8 or exon 7 of the SMN gene in 94% of cases
Homozygous deletion of NAIP gene in 17% of cases
Presence of 3 copies of SMN gene in 54% of cases

Symptoms:

Proximal muscle weakness
Delayed growth and loss of movement skills
Finger tremors
Lack of deep reflexes in tendons

SMA III

In SMA type 3 or Kugelberg-Welander disease, symptoms usually appear over the age of 18 months. This type of SMA does not affect the lifespan of those affected, and in some cases, it has been observed that symptoms do not appear until adulthood.

Cause:

Homozygous deletion of exon 7 or 8 or exon 7 of the SMN gene in 82% of cases
Homozygous deletion of the NAIP gene in 7% of cases
Presence of 4 or more copies of SMN2 gene in 88% of cases signs:  Loss of motor skills

Symptoms:

Loss of movement skills
Difficulty running, walking or climbing stairs due to proximal muscle weakness
Finger tremors
Feeling tired
Recurrent respiratory system infections

SMA IV

This form of SMA is typically experienced by adults, and its mild symptoms do not impact an individual’s ability to live longer. This is the rarest type of SMA, with less than 5% of patients having this condition.

Cause:

Homozygous deletion of exon 7 or 8 or exon 7 of the SMN gene in 0.3% of cases
Homozygous deletion of NAIP gene in less than 2% of cases
Presence of 4 or more copies of SMN2 gene in 88% of cases

Symptoms:

Feeling tired
Weakness in the deltoid, triceps and quadriceps muscles
Decreased reflexes of tendons in upper limbs and Achilles
Shaking hands

SMA prevalence rate

This disease is present in approximately 1 person per 6,000 to 10,000 people worldwide. Sources with reliable data indicate that around 10,000 to 25,000 Americans have some form of SMA, and it is estimated that 1 out of every 54 individuals in the United States has a mutation or deletion of the SMN1 gene. In the following table, the occurrence rate of carriers and individuals impacted in various populations is specified.

Estimated Incidence	Carrier Frequency	Carrier Frequency
Not reported	1:59	The Arabs
1:8009	1:48	Asians
1:9655	1:71	Asian Indian
1:18808	1:100	Black (sub-Saharan African heritage)
1:7829	1:45	White
1:20134	1:77	Hispanic
1:10000	1:56	Jewish

Other names of disease

5q SMA
Proximal SMA
SMA
SMA-associated SMA
Spinal amyotrophy
Spinal muscle degeneration
Spinal muscle wasting

The Cause and inheritance pattern of SMA disease

SMA disease is a genetic disorder that arises from a homozygous loss mutation of the SMA1 gene in 95% of cases. In the remaining 5% of patients, a homozygous deletion mutation in exon 7 of the SMN1 gene is observed, with most of them being heterozygous compounds displaying a deletion mutation in one allele and a point mutation in the other. The mode of inheritance for this disease is autosomal recessive, thus parents who carry the disease have a 25% chance of having an affected child. In individuals with severe SMA, mutations in the SMN2 gene have also been noted. The SMN2 gene acts as a modifier of disease severity by producing a protein product similar to that of the SMN1 gene.

Consequently, a higher number of SMN2 gene copies in affected individuals correlates with a lower severity of symptoms. Therefore, if mutations in both genes are present in a person’s genome, the disease symptoms will be more severe and the life expectancy of SMA patients will decrease. The table below provides a summary of the number of SMN2 gene copies in different types of SMN disease.

SMA III/IV	SMA II	SMA I	SMN2 Copy Number
0%	4%	96%	1
5%	16%	79%	2
31%	54%	15%	3
88%	11%	1%	4=<

In general, the diagnosis of Spinal Muscular Atrophy (SMA) involves the examination of three genes, namely SMN1, SMN2, and NAIP, which are included in the SMA diagnostic kit known as Trita® SMA Diagnostic Kit. All three genes are situated in the 5q13.2 region. The SMN1 gene is specifically located in a duplicated region spanning approximately 500 kb, where a total of five genes SERF1, NAIP, SMN1, SMN2, and GTF2H2 are duplicated. Each gene in this region has both a telomeric and a centromeric copy.

Significantly, the SMN2 gene corresponds to the centromeric version of the SMN1 gene, exhibiting a difference of only five nucleotides in their 3′ region. A specific mutation, known as the c.840 C>T substitution mutation, occurs in exon 7 of the SMN2 gene resulting in alternative splicing in this particular exon. As a consequence, a transcript lacking exon 7 (Δ7-SMN2) is produced. This truncated protein is highly unstable and is prone to quick degradation. Other identified mutations include missense mutations c.689 C > T and c.844 C > T in the SMN1 gene.

The NAIP gene, found in the 5q13 region (5q12.2-q13.3), codes a neuron apoptosis inhibitory protein. This protein plays a crucial role in modulating the differentiation and survival of nerve cells, particularly motor neurons. Despite extensive investigation, the precise contribution of NAIP function to the pathogenesis of spinal muscular atrophy (SMA) remains partially elusive. Nevertheless, certain reports have established a correlation between deletion mutations in specific regions of the NAIP gene (exons 4, 5, and 13) and the severity of SMA disease. Adjacent to the SMN gene, it is hypothesized that this gene rectifies the spinal muscular atrophy phenotype arising from mutations in the neighboring gene, SMN1.

Notably, the centromeric NAIP gene variant, NAIPψ, is characterized by the absence of coding exons 5 and 6. The results of studies have consistently demonstrated a high prevalence of NAIP gene mutations in individuals diagnosed with SMA type 1 (SMA I). Moreover, the complete deletion of this gene significantly accelerates the deterioration of respiratory function in SMA I patients (in comparison to SMA I patients lacking NAIP gene mutations). These patients typically die due to the disease before reaching the age of 6 months. However, if they manage to survive, they will necessitate ongoing artificial respiration support.

The findings of a study conducted on 186 Spinal Muscular Atrophy (SMA) patients in Iran, which was documented in the year 2023, demonstrate that all of the subjects examined displayed a homozygous deletion mutation within exon 7 of the SMN1 gene. Furthermore, it was noted that approximately 90% of individuals affected by this condition displayed a homozygous deletion mutation in exon 8 of the SMN1 gene. In addition, a further outcome disclosed in this investigation is the copy number of SMN2 and NAIP E5 genes within SMA patients, with corresponding graphical representations presented in the subsequent section.

The right picture shows the percentage of people with the copy number of NAIP gene and the left picture shows the percentage of people with different copy numbers of exon 7 of SMN2 gene.

Gene GTF2H2 (transcription factor IIH subunit 2) as well as SERF1 (Small EDRK-Rich Factor 1A) are neighboring genes in close proximity to the SMN gene. The GTF2H2 gene is responsible for encoding a 44 KDa subunit of the transcription initiation factor RNA polymerase II. Although the precise function of the protein produced by the SERF1 gene remains undetermined, it is worth noting that the deletion of the telomeric form of this gene is frequently linked to the deletion of the SMN1 gene in SMA patients. Consequently, it is possible that this gene may serve as a modifier of the phenotype in SMA disorder.

Diagnosis methods

The initial stage of diagnosing SMA disease involves a physical examination and review of the affected person’s medical records by the doctor. Additional tests can be conducted to accurately determine the specific type of the SMA disease upon the attending physician’s preliminary diagnosis, some of which are:

Blood test

In individuals with SMA, the level of creatine kinase enzyme tends to be higher than the normal range due to muscle deterioration.

Muscle biopsy

This procedure entails the extraction of a small amount of muscle tissue for further analysis in a laboratory setting. Muscle sampling biopsy is utilized to diagnose muscle atrophy and examine the composition of muscle tissue.

Nerve and Muscle Testing

The Electromyogram (EMG) or nerve and muscle test is a diagnostic method employed in SMA disease. It involves assessing the level of nerve activity in an individual’s muscles.

Amniocentesis and chorionic villus sampling (CVS)

Both amniocentesis and chorionic villus sampling (CVS) are diagnostic methods utilized during prenatal screenings and throughout pregnancy.

Genetic testing

Considering the significant pain associated with SMA disease and the high costs of treatment, genetic diagnostic tests can be carried out at two distinct levels. The first level involves genetic testing of the parents to determine the probability of them being carriers. The second level encompasses prenatal screenings that evaluate the clinical status of the fetus through precise genetic tests like the MLPA test. In cases where there is a familial history of SMA, it is advisable to undergo genetic testing during prenatal screenings, as the likelihood of a child being born to a carrier parent is 25%.

Today, there exists a range of genetic tests available for the diagnosis of spinal muscular atrophy (SMA), one such method being the Multiplex Ligation-dependent Probe Amplification (MLPA) technique. MLPA is a multiplex Polymerase Chain Reaction (PCR) method that utilizes up to 40 distinct probes, each designed to target a specific sequence. This method offers several notable features, including the capability to assess copy number variations in multiple human genes, detect deletions or duplications in the genetic material and identify alterations in methylation patterns.

The MLPA method employs labeled probes that are synthesized as two half-probes, namely the 5′ MLPA probe and the 3′ MLPA probe. These probes are specifically designed to identify particular sequences. Additionally, MLPA utilizes certain universal primers, enabling the simultaneous multiplex PCR amplification of all probes. It is important to note that this method has both limitations and advantages, which will be discussed further.

In 2013, a study was carried out to evaluate the diagnostic capabilities of MLPA and Real-Time PCR techniques in identifying SMA type 1. A total of 1016 patient samples were utilized in this research. It was observed that a small fraction of the samples, specifically 5 in number (0.5% of the total), were unable to undergo analysis on both platforms. This suggests that the extracted DNA quantities from these particular samples were likely insufficient. The outcomes of this investigation demonstrated that both methodologies possess the ability to accurately ascertain the number of copies of exon 7 of the SMN1 gene.

Nonetheless, there were certain issues encountered with the reproducibility of the MLPA assay in 7 cases, even though both techniques were able to accurately determine the number of SMN2 gene copies in 99% of the samples. In 6 out of the 7 problematic samples, the MLPA technique reported an excess number of replicates (3 replicates instead of 2). These findings highlight the fact that both RT-PCR and MLPA methods are considered valid for determining the copy number of exon 7 of the SMN1 gene and can be employed for population-based carrier screening.

Advantages of the MLPA method

Gold standard method for CNV diagnosis at exon level (along with some microarrays)
Robust, rapid and affordable method
High resolution, accurate detection of exon deletion/duplication mutation
It can be designed for specific single nucleotide variants (SNVs)
The possibility of changing the method in order to detect methylation changes(MS-MPA)

Limitations of the MLPA method

Unable to recognize balanced rearrangement
Lack of commercial MLPA kit for all genes
Possibility of missing small intronic mutations outside the regions studied with the kit’s probes
Inability to recognize the location or direction of the doubled area
Potential failure to detect mosaicism
Failure to detect SNVs (Single Nucleotide Variants) unless it is specifically the target sequence of MLPA probes.
Limitation in the number of target regions in each kit
If the deletion or duplication region extends beyond the target region of the kit probes, it becomes necessary to employ a second method such as microarray analysis.
Observation of false positive results can occur as a consequence of alterations in the sequence of the probe or ligation site, particularly in relation to the BRCA1 gene.
Observation of false positive results during molecular diagnosis of Duchenne/Baker muscular dystrophy due to sequence change (the presence of different types of sequences in individuals of the same population) and as a result of probe hybridization sites.

The MLPA method is generally conducted through a series of five distinct steps, which are:

DNA denaturation and incubation with MLPA probes:

During this phase, the two strands of DNA are separated, allowing the half probes to identify the target sequence. If the probe connection is executed accurately without any gaps, subsequent steps involving ligation and amplification can be carried out.

Ligation
Performing PCR reaction and fragment amplification
Separation of PCR products using capillary electrophoresis under denaturing conditions
Analyzing the results by examining the peak height or the area of the fluorescence peaks, which can be achieved using software such as GeneMarker®

The SMA diagnostic kit, manufactured by TritaGene (Trita® SMA Diagnostic Kit) utilizes the novel MFPA (Multiple Fluorescent Probe Amplification) technique in conjunction with specific primers and probes. This kit is capable of precisely ascertaining the quantity of SMN1 and SMN2 gene copies located in the 7th exon region. These capabilities make it an invaluable tool for the precise diagnosis of this particular disease. It is important to note that the MFPA technique is akin to MLPA, with the exception that the ligation step has been omitted.

Real-time PCR is one of the useful molecular diagnostic methods that can be used for SMA diagnosis. Real-time PCR, also known as quantitative PCR or qPCR, is a form of multiplex PCR that enables the monitoring and observation of reaction progress. This method boasts several advantages, such as its rapidity, precision and heightened sensitivity, which have established it as a crucial tool in disease diagnosis.

Notably, the quantity of primary DNA sample present determines the number of amplicons in the PCR reaction, presenting a key consideration in real-time PCR. Additionally, this technique finds important applications in gene expression analysis, wherein mRNA is initially transformed into cDNA through reverse transcriptase. Subsequently, cDNA serves as a template in the real-time PCR reaction facilitated by labeled probes and primers. The replication of the relevant sequence is explored, and these probes are designed to emit light that correlates with the progression of reaction stages and product generation. Within the real-time PCR technique, the term “Ct” or threshold cycle denotes the initial cycle in which fluorescent light production exceeds the first phase (Linear ground phase).

The results of Real-Time PCR are presented in the form of graphs concurrently with the advancement of the reaction process, and these graphs exhibit the four principal stages of the reaction. The various stages include:

The initial or Linear ground phase

During this stage, the components are being rapidly synthesized, although the signal is not sufficiently robust to be detected by the apparatus.

The second or Early exponential phase

During this stage, the amplification is performed exponentially, and the apparatus receives the corresponding signals.

The third or Log-linear phase

In this stage, the pace of the Real-Time PCR reaction is gradually diminished and the reaction materials and their efficacy are reduced.

The fourth or Plateau phase

In the fourth stage, no alteration in the level of fluorescent light detected by the apparatus is observed, which signifies the depletion of the compounds in the reaction.

The Real-Time PCR technique typically employs two types of fluorescent dyes. The first type consists of fluorescent dyes such as SYBR Green and EvaGreen, that have the capacity to bind to any double-stranded DNA present in the reaction medium in a non-specific manner. The second group of fluorescent dyes is linked to oligonucleotide sequences referred to as probes. These dyes are capable of exclusively binding to specific sequences, including hydrolytic probes like TaqMan probes and beacons.

Treatment

Can SMA be cured? Despite the known genetic cause of SMA disease, a definitive treatment for it is currently unavailable. Only certain drugs have the potential to restore the SMN1 gene or increase the expression of the SMN2 gene, thereby compensating for the FL-SMN protein. In the subsequent sections, we will introduce several of these drugs.

Nusinersen (Spinraza), developed in partnership with Biogen, stands as the initial medication approved by the US Food and Drug Administration (FDA) in 2016 for the treatment of SMA patients. This drug can be administered to patients of all ages and consists of a 2′-O-methoxyethyl antisense oligonucleotide (ASO) that aims to enhance the expression of the SMN protein. Nusinersen’s mechanism of action involves binding to the intron-splicing silencer region N1 in SMN2 pre-mRNA, leading to the creation of exon 7 and subsequently enabling SMN2 to produce FL-SMN protein. It is important to note, however, that this drug cannot traverse the blood-brain barrier and must therefore be administered into the spinal cord.

AVXS-101 (Zolgensma) is an additional medication employed for patients with Spinal Muscular Atrophy (SMA), that was developed by AveXis in 2019 and obtained approval from the Food and Drug Administration (FDA). AVXS-101 is a non-replicating recombinant Adeno-Associated Virus serotype 9 (AAV9) that possesses DNA which is complementary to the human Survival Motor Neuron (SMN) gene under the influence of the cytomegalovirus enhancer/chicken-β-actin-hybrid promoter. Administering this medication allows patients to attain systemic and long-lasting expression of the SMN1 gene, thereby enhancing their motor capabilities.

Risdiplam (Evrysdi) produced by Genentech, a subsidiary of the Roche group, received FDA approval in 2020. This therapeutic intervention can be administered orally to individuals ranging from children over the age of 2 months to adults. Risdiplam is an mRNA splicing modifier that elevates the expression of the SMN protein in individuals affected by SMA.