Spinal muscular atrophy (SMA) is one of the most common autosomal recessive neuromuscular disorders affecting infants and children. SMA incidence was estimated to be 1 in 6000–10,000 live births worldwide with a carrier frequency of 1:40–80 among different ethnic groups [5, 7]. The highest reported prevalence of disease-prone mutation carrier is among European Caucasoids with 1/47 frequency, followed by 1/52 in Asian Indians, 1/59 in Asians, 1/68 in Hispanics, and 1/72 in African Americans [32, 33].
SMA is a neuromuscular disorder resulting from the irreversible degeneration of the anterior horn cells of the α-motor neurons of the spinal cord, producing a proximal progressive muscles atrophy, and may lead to paralysis. Respiratory muscle weakness together with the thoracic cage deformity frequently results in respiratory failure and death, particularly in severe cases or with early-onset patients [3].
Clinical phenotypes of SMA have a heterogeneous range from a severe to a mild phenotype. It is now classified into five subtypes (types 0 to IV) based on the age of onset and severity of the condition. SMA type 0 is the most severe form with uterine onset, and death usually occurs before six months of age. Type I (Werdnig-Hoffmann disease, OMIM# 253300) represented the most common subtype in over half of the reported SMA cases with muscle weakness persisting at birth or before six months of age and patients usually die of respiratory failure within two years. Type II (OMIM# 253550), has an onset usually 18 months after birth, with patients able to sit but never walk by themselves and can survive beyond four years of age. The late-onset types are type III (Wohlfart–Kugelberg–Welander disease, OMIM#253400), in which the onset is delayed to more than 18 months and patients are able to walk but often become wheelchair-bound during youth or adulthood), [5, 33], while SMA type IV is the mildest late-onset form with normal life expectancy [32].
The SMA determining gene is called the “survival motor neuron” gene (SMN, OMIM #600354, #601627) located on 5q13. The large inverted duplication consists of two homologous genes arranged in tandem on each chromosome; SMN1 (telomeric copy, the disease-causing gene) and SMN2 (paralog centromeric copy). Both genes consist of nine exons and share more than 99% nucleotide identity with exon 8 remaining untranslated. They differ only by five single nucleotide variants (SNVs) within their 3′ ends; two SNVs are located in the coding region of exons 7 & 8, one in intron 6 and two in intron 7. These five unique SNVs are used as a diagnostic tool that allows the distinction between SMN1 and SMN2 genes [1, 7, 10].
Although both genes produce equal transcript amounts, almost 70–85% of SMN2 derived transcripts are unstable, truncated and not fully functioning due to the exon 7 nucleotide exchange NM_000344.3:c.840C > T that interrupts a splicing enhancer and results in exon 7 skipping [33]. SMN2 gene partially compensates the SMN protein with small amount of functional protein, thus an inverse correlation arises between the number of SMN2 copies and the severity of the disease. SMA patients commonly have at least one SMN2 copy. Carriers on the other hand are asymptomatic because they retain one functioning copy of SMN1 gene but can pass their nonfunctioning copy to their children [3, 4, 16].
In this highly homologous region, a gene conversion between SMN1 and SMN2 can occur producing a hybrid SMN gene. Gene conversion mainly takes place by fusion of SMN1 exon 8 with SMN2 exon 7 converting SMN1 into SMN2 or vice versa. This results in variable copy numbers (CNs) of SMN1 and SMN2 [5, 27].
Approximately 95% of SMA patients are due to homozygous deletion of exon 7 of SMN1 gene. The remaining 5% shows other pathogenic point mutations in compound either in homozygous form or in compound heterozygosity with SMN1 deletion. Since the disease is autosomal recessive; de-novo variants are causative reasons in only 2% of the affected patients [21].
Three early treatments, Spinraza®(nusinersen), Zolgensma® (onasemnogene abeparvovec-xioi, OA), and Evrysdi® (risdiplam) received FDA approval for the amelioration of SMA symptoms and enhancing of long-term quality of life and survival [5, 11].
Spinraza® (Biogen, Cambridge, MA, USA) is the first SMA effective treatment and is a modified antisense oligonucleotide-based therapy that enhances the production of SMN protein by increasing the production of full-length SMN proteins [11, 21] .
Zolgensma® (onasemnogene abeparvovec “OA”) is a gene replacement therapy that delivers a cDNA coding for the SMN protein using Adeno-associated virus 9 (AAV9) as a vector. It is systemically applied to children less than 2 years employed at two different doses [14].
Evrysdi® is an oral SMN2 pre-mRNA splicing modifier recently approved for the treatment of SMA patients aged 2 months and older. Risdiplam directly promotes the generation of full-length SMN2 mRNA—which increases the production of functional SMN protein [23].
SMA treatment in pre-symptomatic infants increased the likelihood of survival and improved motor function. For an early diagnosis and the initiation of treatment, newborn SMA screening, carrier frequency of SMA, and SMN2 copy number are important [21, 34].
The comparative Ct (threshold cycle) method can be used to determine the copy number of SMN1 gene by simple quantitative real-time PCR assays. It detects the most common mutation in SMA and approximately 90% of carriers. This test can be used for Genetic counseling in such patients to reduce the likelihood of having an affected child in the future [1, 10].
This study is a pilot study aiming at throwing some light on the importance of SMA carrier detection in Egypt. Herein, we report the results of qPCR quantification of the SMN1 gene in geographically heterogeneous Egyptian samples, as well as in SMA families seeking genetic counseling.
