Fang Song

Background: Spinal muscular atrophy (SMA) is mainly caused by biallelic SMN1 inactivation. While most patients carry homozygous deletions, 3–5% are compound heterozygotes, making molecular diagnosis challenging. Methods: A tiered diagnostic strategy was applied to 17 pediatric patients, combining copy number analyses (MLPA and targeted long-read sequencing, tLRS), sequence variant detection (RT-PCR cloning and sequencing, allele-specific long-range PCR with nested PCR, and tLRS), and structural variant analysis (ultra-long-read sequencing, Ultra-LRS). Results: Copy numbers were concordant between MLPA and tLRS. MLPA-suggested gene conversions were confirmed by tLRS, while discordant total copy numbers were resolved as large deletions by Ultra-LRS. RT-PCR cloning, and sequencing identified SMN1 variants in 11/12 cases and confirmed aberrant splicing in three cases, but failed for large deletions. AS-LR-PCR with nested PCR characterized the variants in 13/15 but failed in gene conversion cases. tLRS achieved definitive diagnosis in all cases, and Ultra-LRS precisely delineated breakpoint junctions of two large deletions. Conclusions: A hierarchical complementary strategy integrating copy number, sequence, and structural analyses is essential for the accurate diagnosis of compound heterozygous SMA.

Background Biomarkers can be used to assess the severity of spinal muscular atrophy (5q SMA; SMA). Despite their potential, the relationship between biomarkers and clinical outcomes in SMA remains underexplored. This study aimed to assess the association among biomarkers, phenotypes, and motor milestones in Chinese patients diagnosed with SMA. Methods We collected retrospective clinical and follow-up data of disease-modifying therapy (DMT)-naïve patients with SMA at our center from 2019 to 2021. Four biomarkers were included: survival motor neuron 2 (SMN2) copies, neuronal apoptosis inhibitory protein (NAIP) copies, full-length SMN2 (fl-SMN2), and F-actin bundling protein plastin 3 (PLS3) transcript levels. Data were analyzed and stratified according to SMA subtype. Results Of the 123 patients, 30 were diagnosed with Type 1 (24.3%), 56 with Type 2 (45.5%), and 37 with Type 3 (30.1%). The mortality rate for Type 1 was 50%, with median survival times of 2 and 8 months for types 1a and 1b, respectively. All four biomarkers were correlated with disease severity. Notably, fl-SMN2 transcript levels increased with SMN2 copies and were higher in Type 2b than those in Type 2a (p = 0.028). Motor milestone deterioration was correlated with SMN2 copies, NAIP copies, and fl-SMN2 levels, while PLS3 levels were correlated with standing and walking function. Discussion Our findings suggest that SMN2 copies contribute to survival and that fl-SMN2 may serve as a valuable biomarker for phenotypic variability in SMA Type 2 subtypes. These insights can guide future research and clinical management of SMA.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by homozygous deletions or mutations in survival motor neuron gene 1 (SMN1). Currently, the primary therapeutic strategy for SMA is to increase the level of SMN via correcting SMN2 splicing (nusinersen and risdiplam). However, some patients with SMA do not respond to such treatments, thereby warranting a need to develop new therapeutic strategies. We have previously reported that SMN2 expression is epigenetically regulated by DNA methylation levels of the SMN2 promoter region. In the present study, we determined that methyl-CpG-binding protein 2 (MeCP2) may bind to this critical promoter region (nt-167 to 43). Antisense oligonucleotides (ASO-P1 and ASO-P2) were designed to target the key methylation sites in the SMN2 promoter region, which enhanced the overall transcription and functional protein expression levels in the SMA cell lines. These results were similar to those observed in nusinersen-treated SMA cells. Moreover, a combined treatment of ASO-P1 and ASO-NUS in SMA cell lines further increases fl-SMN2 transcript and SMN protein levels. The delivery of ASO-P1 to the central nervous system of severe SMA mice corrected the molecular, pathological, and functional phenotypes of this disease and increased survival rates. Our findings suggest that the key methylation regions in the SMN2 promoter region may be a novel therapeutic target for SMA.

Background: Patients with spinal muscular atrophy (SMA) are at risk of decreased bone mineral density (BMD). The bone health status of Chinese patients with SMA has been poorly studied. We aimed to evaluate the BMD of children with SMA types 2 and 3 in mainland China and investigate its influencing factors. Methods: Forty patients with a mean age of 5.5 years affected by SMA types 2 and 3 (n = 22 and n = 18, respectively) were enrolled between September 2017 and May 2019. Total body less head (TBLH) BMD, lumbar spine (LS) BMD, and body composition were measured using dual-energy X-ray absorptiometry (DXA). Serum bone metabolism markers and complete spinal radiographs were assessed. We utilized a linear regression model to explore the correlations between BMD and its related factors. Results: A total of 67.5% (27/40) of patients were diagnosed with low BMD and 2.5% (1/40) were diagnosed with osteoporosis. The TBLH BMD and LS BMD Z-scores in children with SMA type 2 were significantly lower than those with SMA type 3. Both TBLH and LS BMD Z-scores tended to increase with the change of SMA subtypes from 2a-3b. Vitamin D insufficiency and deficiency were found in 37.5% (15/40) of the patients. Serum Ca, phosphorus (P), alkaline phosphatase (ALP) and parathormone (PTH) levels were normal. There were no significant differences among the four subtypes in terms of all the serum bone metabolism markers. Phenotype was significantly associated with TBLH BMD and LS BMD Z-scores, and serum PTH levels were significantly associated with TBLH BMD Z-scores. Conclusions: Low BMD and osteoporosis were highly prevalent in mainland Chinese children with SMA types 2 and 3. Phenotype and serum PTH level might be the influencing factors of BMD. Regular monitoring of BMD by DXA scan and taking active interventions aim to SMA children with different types are important.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by deletion or subtle variant of survival motor neuron 1 (SMN1) gene. By multiplex ligation-dependent probe amplification, genomic sequencing, and T-A cloning on cDNA level, we identified one novel SMN1 subtle variant c.835G>C (p.Gly279Arg) in a non-homozygous patient with type 1 SMA. Full-length SMN1 (fl-SMN1) transcripts in the peripheral bloods of the patient were significantly decreased compared with those in healthy individuals and the carries (p < 0.05). And two fragments of SMN1 transcripts including fl-SMN1 and △7-SMN1 were observed by RT-PCR, which indicated Exon 7 skipping of SMN1 gene. To further evaluate its splicing effects on Exon 7, we performed ex vivo splicing analysis, which showed that the mutant mini gene with c.835G>C reduced Exon 7 inclusion to 54%. In addition, self-oligomerization between mutant SMN protein with the c.835G>C (p.Gly279Arg) and wild SMN was decreased in self-interaction assays. Our study clearly demonstrates that the c.835G>C (p.Gly279Arg) variant can lead to a decrease in fl-SMN1 transcripts by interrupting correct splicing of SMN1. What is more, the variant also affects SMN self-oligomerization via amino acid substitution from Gly to Arg at amino acid position of 279. This work presents the first evidence that it does exit double-hit events for the novel variant, which is crucial to understanding a severe SMA phenotype (type 1).

To define the relationship between the survival motor neuron 1 gene (SMN1) and SMN2, and explore the variability of these two genes within the generations, SMN1 and SMN2 copy numbers were determined for 227 SMA families. The association analysis indicated that there was a negative correlation between the copy number of SMN1 and SMN2 (Spearman = −0.472, P < 0.001) in 227 SMA children and 454 of their parents. The average SMN copies from father and mother in each SMA family were used to represent the copy number in the parent’s generation. Subsequently, SMN transmission analysis showed that the similar distribution trend of SMN1 and SMN2 copy number was not only in the SMA children and their parents’ generation but also in the non-SMA families. Moreover, when the SMN2 copy number was one in the parent’s generation, 75% of their SMA children had type I and 25% of them had type II/III. However, when the SMN2 copies were three in the parent’s generation, all of their SMA children were type II/III. Therefore, the diversity of SMN copies was mostly inherited and the SMN2 copy number in the parent’s generation could predict the disease severity of SMA children to some extent.