Chinese Spinal Muscular Atrophy Research Articles

Rehabilitation for spinal muscular atrophy patients in China: a national cross-sectional study

BackgroundThe management of Spinal Muscular Atrophy (SMA) requires a multidisciplinary treatment approach, wherein rehabilitation constitutes an integral element. In this study, we examined the effects of rehabilitation among Chinese SMA patients and assessed the real-world efficacy of rehabilitation interventions.MethodsWe conducted a cross-sectional online survey on SMA patients from June 9, 2023, to June 30, 2023, through the Meier Advocacy & Support Center using data from the Center’s database and electronic questionnaires. The rehabilitation situation of the participants over the past 14 months were investigated. Logistic binary regression was used to analyze the relationship between Pediatric Quality of Life Inventory(PedsQL™) scores and rehabilitation.ResultA total of 186 questionnaires were finally analyzed. Only 29 patients did not rehabilitated in the past 14 months. A significant correlation between age and type of rehabilitation, as well as between age and duration of rehabilitation. Patients receiving no rehabilitation or solely home-based rehabilitation exhibited a higher median age of 8.4 compared to those undergoing standard rehabilitation or a combination of standard and home-based rehabilitation, with a median age of 4.9 (z-score = -4.49, p-value < 0.001). In addition, long-term rehabilitation (OR = 0.314, 95%CI = 0.106–0.927, p = 0.04) were negatively correlated with lower PedsQL™ Neuromuscular Module scores, and PedsQL scores in the long-term rehabilitation group were higher than those in the short-term and no-rehabilitation groups (54.2 ± 15.1 vs. 45.9 ± 14.4 and 42.3 ± 14.3, p = 0.01), with the most significant difference observed in the physical function section (59.0 ± 15.8 vs. 46.8 ± 15.2 and 45.6 ± 15.9, p < 0.01). Mobility and exercise (OR = 0.26, 95%CI = 0.08–0.81, p = 0.02), as well as assistive technology (OR = 0.28, 95%CI = 0.10–0.82, p = 0.02), were independently associated with a lower score in a negative direction.ConclusionThe study found that long-term rehabilitation was linked to higher PedsQL scores in SMA patients, highlighting the need for standardized rehabilitation programs to enhance function and quality of life.

Identification of novel SMN1 subtle mutations using an allelic-specific RT-PCR

Spinal muscular atrophy (SMA) is caused by homozygous deletions of the SMN1 gene in approximately 95% of patients. The remaining 5% of patients with SMA retain at least one copy of the SMN1 gene carrying insertions, deletions, or point mutations. Although molecular genetic testing for most SMA patients is quite easy, diagnosing “nondeletion” SMA patients is still compromised by the presence of a highly homologous SMN2 gene. In this study, we analyzed the SMN1/SMN2 copy number by quantitative PCR and multiplex ligation-dependent probe amplification (MLPA). Further, common primers for both SMN1 and SMN2 sequences were used to screen DNA intragenic mutations. To confirm whether the identified mutations occurred in SMN1 or SMN2, we improved the traditional RT-PCR method by only amplifying SMN1 transcripts using an allelic-specific PCR (AS-RT-PCR) strategy. We identified six SMN1 point mutations and small indels in 8 families, which included c.683T>A, c.22dupA, c.815A>G, c.19delG, c.551_552insA and c.401_402delAG. To the best of our knowledge, the latter three have never been previously reported. The most common mutation in Chinese patients is c.22dupA, which was identified in three families. In this work, we demonstrated AS-RT-PCR to be reliable for identifying SMN1 subtle mutations, especially the prevalent mutation c.22dupA in Chinese SMA patients. By reviewing published papers and summarizing reported SMN1 mutations, a distinct ethnic specificity was found in SMA patients from China. Our research extends the SMN1 mutation spectrum.

The SMN1 common variant c.22 dupA in Chinese patients causes spinal muscular atrophy by nonsense-mediated mRNA decay in humans

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is mostly caused by homozygous deletion of the SMN1 gene. Approximately 5%–10% of SMA patients are believed to have SMN1 variants. c.22 dupA (p.Ser8lysfs*23) has been identified as the most frequent variant in the Chinese SMA population and to be associated with a severe phenotype. However, the exact molecular mechanism of the variant on the pathogenesis of SMA is unclear. We observed that SMN1 mRNA and the SMN protein in the peripheral blood cells of a patient with c.22 dupA were lower than those of controls. The aim of this study is to investigate whether nonsense-mediated mRNA decay (NMD) plays a role in the mechanism of the c.22 dupA variant of the SMN1 gene as it causes SMA. Two lymphoblasts cell lines from two patients (patient 1 and 2) with the c.22 dupA, and one dermal fibroblasts cell line from patient 2 were included in our study. Two-stage validation of the NMD mechanism was supplied. We first measured the changes in the transcript levels of the SMN1 gene by real-time quantitative PCR after immortalized B-lymphoblasts and dermal fibroblasts cells of the SMA patients were treated with inhibitors of the NMD pathway, including puromycin and cyclohemide. Next, lentivirus-mediated knockdown of the key NMD factor—Up-frameshift protein 1 (UPF1)—was performed in the fibroblasts cell line to further clarify whether the variant led to NMD, as UPF1 recognizes abnormally terminated transcripts as NMD substrates during translation. SC35 1.7-kb transcripts, a physiological NMD substrate was determined to be a NMD positive gene in our experiments. The two inhibitors resulted in a dramatic escalation of the levels of the full-length SMN1 (fl-SMN1) transcripts. Additionally, the SC35 1.7-kb mRNA levels were also increased, suggesting that NMD pathway is suppressed by the two inhibitors. For the 3 cell lines, the fold increase of the SMN1 transcript levels of cycloheximide ranged from 2.5±0.4 to 8.3±0.1, 1.9±0.2 to 5.0±0.7 and 2.2±0.1 to 4.9±0.2 for two lymphoblastoid cell lines and one fibroblasts cell line, respectively. For these cell lines, the fold increases of the SMN1 transcript levels of puromycin were as follows: 5.5±0.2 to 19.5±4.0, 3.1±0.3 to 9.9±1.8 and 1.5±0.2 to 6.5±0.5. Meanwhile, the SC35 1.7-kb transcript levels were markedly increased in all 3 cell lines. In addition, lentivirus-mediated UPF1 knockdown lead to a reduction of the UPF1 protein level to 22.5% compared to the negative control lentivirus. Additionally, knockdown of the UPF1 gene also promoted mRNA expression of the SC35 1.7kb and fl-SMN1 genes. The increases of the SMN1 and SC35 1.7-kb mRNA levels reached about 4- and 6.5-fold in fibroblasts derived from the patient 2, respectively. Altogether, our study provides the first evidence that the c.22 dupA variant in the SMN1 gene triggers NMD. SMA pathogenesis in the patient is associated with mRNA degradation of SMN1, but not the truncated SMN protein.

Mutation Spectrum of the Survival of Motor Neuron 1 and Functional Analysis of Variants in Chinese Spinal Muscular Atrophy

Proximal spinal muscular atrophy (SMA) is a common fatal autosomal recessive disorder caused by deletion or mutation of the survival of motor neuron 1 (SMN1). Here, we studied SMA molecular pathology in 653 Chinese patients and found approximately 88.2% with homozygous SMN1 exon 7 deletion and 6.3% with heterozygous exon 7 loss using multiplex ligation-dependent probe amplification. SMN1 variants were detected in 34 patients with heterozygous SMN1 loss by clone sequencing. In 27 of them, 15 variants were identified: five were unreported novel variants [c.-7_9del(p.0), p.Tyr109Cys, p.Ile249Tyrfs*16, p.Tyr272Trpfs*35, and c.835-5T>G], five were previously found only in Chinese patients (p.Ser8Lysfs*23, p.Gln14*, p.Val19Glyfs*21, p.Leu228*, and p.Tyr277Cys), and five were reported in other populations [p.Ala2Gly, p.Gln15*, p.Glu134Lys, p.Ser230Leu, and c.863G>T (r.835_*3del, p.Gly279Glufs*5)]. Variants p.Ser8Lysfs*23 and p.Leu228* were the most common in Chinese SMA. Five variants (p.Ser8Lysfs*23, p.Gln14*, p.Gln15*, p.Val19Glyfs*21, and p.Leu228*) resulted in premature stop codons, likely causing SMN1 mRNA nonsense-mediated decay. The novel variant c.-7_9del (p.0) caused deletion of the translation start codon (AUG), resulting in full-length SMN protein loss. The novel variant c.835-5T>G, located in a splice site, resulted in 90% exon 7 skipping. Our study could facilitate early diagnosis for SMA patients in mutation detection and revealed the specific mutation spectrum ofSMN1 in Chinese SMA and high genetic heterogeneity in subtle variants observed between patients from China and Caucasians.

Molecular characterization and copy number of SMN1, SMN2 and NAIP in Chinese patients with spinal muscular atrophy and unrelated healthy controls.

BackgroundSpinal muscular atrophy (SMA) is caused by SMN1 dysfunction, and the copy number of SMN2 and NAIP can modify the phenotype of SMA. The aim of this study was to analyze the copy numbers and gene structures of SMA-related genes in Chinese SMA patients and unrelated healthy controls.MethodsForty-two Chinese SMA patients and two hundred and twelve unrelated healthy Chinese individuals were enrolled in our study. The copy numbers and gene structures of SMA-related genes were measured by MLPA assay.ResultsWe identified a homozygous deletion of SMN1 in exons 7 and 8 in 37 of 42 patients (88.1%); the other 5 SMA patients (11.9%) had a single copy of SMN1 exon 8. The proportions of the 212 unrelated healthy controls with different copy numbers for the normal SMN1 gene were 1 copy in 4 individuals (1.9%), 2 copies in 203 (95.7%) and 3 copies in 5 (2.4%). Three hybrid SMN genes and five genes that lack partial sequences were found in SMA patients and healthy controls. Distributions of copy numbers for normal SMN2 and NAIP were significantly different (P < 0.001) in people with and without SMA.ConclusionThe copy numbers and gene structures of SMA-related genes were different in Chinese SMA patients and healthy controls.

Molecular analysis of the SMN gene mutations in spinal muscular atrophy patients in China

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive diseases. Survival motor neuron1 (SMN1) is the SMA disease-determining gene. We examined the molecular basis of SMA in 113 Chinese SMA patients. Homozygous exon 7 and 8 deletions in SMN1 were detected by PCR-RFLP. Heterozygous deletion of SMN1 was analyzed based on variation of the sequencing peak height of the two different base pairs of exons 7 and 8 between SMN1 and SMN2. Subtle mutation was detected by genomic sequencing in the patients with heterozygous deletion of SMN1. In our study, the rate of deletion of SMN1 exon 7 and/or 8 was 91.2%; the rate of subtle mutations was 1.8%. We detected the same subtle mutation (p.Leu228X) of SMN exon 5 in two patients (one type I, one type III). The p.Ser8LysfsX23 and p.Leu228X mutations accounted for 13 of the 23 families with subtle mutations reported in the SMN1 gene of Chinese SMA. This is the first report where the phenotype of SMA-type III is associated with p.Leu228X. We found two subtle mutation hotspots (p.Ser8LysfsX23 and p.Leu228X) of SMN1 exons 1 and 5 in Chinese SMA patients. These two mutations have not been reported from America or Europe. It is proposed that the distribution of subtle mutations of SMN1 of SMA is associated with ethnicity or geographic origin.

Subtle mutations in the SMN1 gene in Chinese patients with SMA: p.Arg288Met mutation causing SMN1 transcript exclusion of exon7

BackgroundProximal spinal muscular atrophy (SMA) is a common neuromuscular disorder resulting in death during childhood. Around 81 ~ 95% of SMA cases are a result of homozygous deletions of survival motor neuron gene 1 (SMN1) gene or gene conversions from SMN1 to SMN2. Less than 5% of cases showed rare subtle mutations in SMN1. Our aim was to identify subtle mutations in Chinese SMA patients carrying a single SMN1 copy.MethodsWe examined 14 patients from 13 unrelated families. Multiplex ligation-dependent probe amplification analysis was carried out to determine the copy numbers of SMN1 and SMN2. Reverse transcription polymerase chain reaction (RT-PCR) and clone sequencing were used to detect subtle mutations in SMN1. SMN transcript levels were determined using quantitative RT-PCR.ResultsSix subtle mutations (p.Ser8LysfsX23, p.Glu134Lys, p.Leu228X, p.Ser230Leu, p.Tyr277Cys, and p.Arg288Met) were identified in 12 patients. The p.Tyr277Cys mutation has not been reported previously. The p.Ser8LysfsX23, p.Leu228X, and p.Tyr277Cys mutations have only been reported in Chinese SMA patients and the first two mutations seem to be the common ones. Levels of full length SMN1 (fl-SMN1) transcripts were very low in patients carrying p.Ser8LysfsX23, p.Leu228X or p.Arg288Met compared with healthy carriers. In patients carrying p.Glu134Lys or p.Ser230Leu, levels of fl-SMN1 transcripts were reduced but not significant. The SMN1 transcript almost skipped exon 7 entirely in patients with the p.Arg288Met mutation.ConclusionsOur study reveals a distinct spectrum of subtle mutations in SMN1 of Chinese SMA patients from that of other ethnicities. The p.Arg288Met missense mutation possibly influences the correct splicing of exon 7 in SMN1. Mutation analysis of the SMN1 gene in Chinese patients may contribute to the identification of potential ethnic differences and enrich the SMN1 subtle mutation database.

Analysis of survival motor neuron gene conversion in patients with spinal muscular atrophy

To investigate the type and frequency of gene conversion from SMN1 to SMN2 in Chinese patients affected with spinal muscular atrophy (SMA), and to explore the relationship between gene conversion and clinical phenotype. Non-homozygous deletion of SMN1 gene exon 8 was screened among 417 patients with SMN1 exon 7 homozygous deletions. To analyze and verify the types of gene conversion, genomic DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), and gene subcloning and sequencing were carried out. Thirty-one patients (7.4% of all) with non-homozygous deletions of SMN1 exon 8 were detected. Through series of experiments, the fusion genes SMN1/SMN2 in all cases were delineated. Five types of gene conversions were identified, which included SMN2-I7b/SMN1 E8, SMN2-I7a/SMN1 I7b, SMN2-E7/SMN1 I7a, SMN1 I6/SMN2 E7/SMN1 I7a and SMN2-E7/SMN1 I7a/SMN2 I7b. Such conversions were found in the type I-III patients. For 10 patients with type I-III SMA and 3 copies of SMN2 gene produced by conversion, the average survival age was 5 year and 4 months. Partial conversions of SMN1 gene have been found among Chinese SMA patients. The type of conversion and frequency seem to be different from those of other races. Gene conversion to some extent may impact on survival time and rate of SMA patients, especially type I SMA.

Mutation analysis of SMN1 gene in patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder. About 80% - 90% of SMA patients are missing both copies of SMN1, and 5% - 10% of patients are compound heterozygotes. In the present study, we aimed to analyze survival motor neuron 1 (SMN1) gene mutation in three patients with spinal muscular atrophy and their families to explore the effect of mutation on SMN protein function and the relationship between mutation and clinical phenotype. According to the international criterion, all patients were diagnosed by a neurologist. Patient 1 is a 5 years old boy with SMA type II. Patient 2, female, 2.5 years old, was SMA type II. Patient 3, female, 9 years old, was SMA type III. The brother of patient 3 was SMA type II, too. The age at last examination was 14 years. Genomic DNA was extracted from peripheral blood leukocytes by using standard phenol/chloroform method and total RNA was extracted from whole blood with QIAamp RNA Blood Mini Kit. PCR/RFLP was used to detect the homozygosis deletion of the SMN1 exon 7, and multiplex ligation-dependent probe amplification (MLPA) were performed to analyze the gene dosage of SMN1 and SMN2 for each patient and his/her family members; reverse transcriptase (RT)-PCR and clone sequencing were conducted for identifying the point mutation of SMN1 in three patients. The sequencing of genomic DNA and MLPA were carried out in the 3 families members to confirm the transition of mutation. No homozygous deletion of the SMN1 exon 7 was observed in any member of the 3 families. Case 1 and case 2 had one SMN1 copy compound with c.400G > A (p.Glu134Lys) mutation on it and SMN2 was two copies, respectively. Case 3 and her brother also had one copy of SMN1 and two copies of SMN2, and a mutation c.689C > T (p.Ser230Leu) occurred on the retained SMN1. All point mutations were from their fathers and deletion come from their mothers for SMN1 gene. In this work, p.Glu134Lys and p.Ser230Leu mutations were identified in three unrelated families and p.Glu134Lys from two patients was first discovered in Chinese SMA. The p.Glu134Lys mutation within the SMN Tudor domain prevents the binding of SMN and Sm. The fact that p.Ser230Leu results in a polar amino acid substituted for non-polar amino acid possibly affects the structure of SMN and then damages its function. SMN1 point mutation analysis is not only advantageous to the diagnosis of those patients with heterozygous deletion of SMN1, but will be beneficial to the prenatal diagnosis and genetic counseling for their families.

Point mutation analysis of SMN1 gene in patients with spinal muscular atrophy

To identify the point mutations in survival motor neuron gene 1 SMN1 gene and confirm the existence of compound heterozygous mutations in Chinese patients with spinal muscular atrophy (SMA). Three unrelated patients were diagnosed and clinically typed according to the criteria of proximal SMA established by the International SMA Consortium. Multiplex ligation-dependent probe amplification (MLPA) analysis was carried out to measure the copy numbers of SMN1, SMN2 and neuronal apoptosis inhibitory protein gene (NAIP)in the patients. The point mutation analysis of SMN1 gene was performed by reversed transcript-polymerase chain reaction (RT-PCR) and cloning sequencing. The MLPA assay and point mutation analysis were also performed in the family members to confirm the transmission of the mutations. Two point mutations were identified in the present study, i.e., the p.Leu228X in one patient and p.Arg288Met in two patients. The mutation p.Arg288Met was first reported in Chinese and p.Leu228X was first reported in Mainland Chinese. The case carrying p.Leu228X mutation was diagnosed as SMA I with 2 copies of SMN2, and the cases with p.Arg288Met were diagnosed as SMA I and SMA II , respectively, with 3 copies of SMN2 gene. The mutations p.Leu228X and p.Arg288Met caused severe clinical phenotypes, SMA I or SMA II. This study suggested that the compound heterozygous mutations of SMN1 existed in Chinese SMA patients, which was rarely reported previously in Chinese. It was necessary to detect the point mutation in SMN1 for genetic diagnosis of those patients with heterozygous deletion of SMN1, which would be beneficial to prenatal diagnosis and genetic counseling in these families.

Evaluation of an in-house protocol for prenatal molecular diagnosis of SMA in Chinese

Background Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of the anterior horn of the spinal cord, leading to symmetric muscle weakness and atrophy. About 95% of SMA patients have homozygous loss of SMN1 which can be detected by conventional PCR-RFLP testing. However, the method cannot distinguish heterozygous healthy carriers. A quantitative method named multiple ligation-dependent probe amplification (MLPA) was introduced in our protocol for prenatal molecular diagnosis of SMA in our laboratory. Methods DNA was extracted from amniotic fluid cells of 13 fetuses from 11 Chinese SMA families. STR profiling was carried out to evaluate the contamination of amniotic DNA by maternal genomic DNA. Three methods, PCR-RFLP, allele-specific PCR and MLPA, were used to analyze SMN1 exon 7 of amniotic DNA. Results There was no contamination of amniotic DNA by maternal genomic DNA. In conventional PCR-RFLP, allele-specific PCR, and MLPA, homozygous loss of SMN1 was observed in 4 fetuses. Among the remaining 9 fetuses, 6 with 1 copy of SMN1 and 3 with 2 copies of SMN1 were showed by MLPA. Conclusion This in-house protocol was reliable and efficient for prenatal molecular diagnosis of SMA.

Molecular analysis of SMN, NAIP and P44 genes of SMA patients and their families

Mutations of the telomeric survival motor neuron gene (SMN1) are related to spinal muscular atrophy (SMA). However, no phenotype–genotype correlation has been observed since the SMN1 gene is lacking in the majority of patients affected with either the severe form (type I) or the milder forms (types II and III). Here, we analyze the SMN, NAIP and P44 genes in 132 Chinese SMA patients and their families. At least three types of normal allele, and four types of mutant allele were found in this study. The combination of one normal allele with one mutant allele resulted in carriers of different types, and the combination of different mutant alleles accounted for the different genotypes among different types of SMA. Deletions of mutant alleles can be further subgrouped into four types, which includes involving SMN1, SMN1 and NAIP T (telomeric portion of NAIP gene), SMN1 and NAIP T and P44 T (telomeric portion of P44 gene), and SMN1 and SMN2 (centromeric portion of SMN gene). Some of the severe (type I) SMA cases correlated with the extent of deletions in the SMN, NAIP and P44 genes or the dosage of SMN gene when both SMN1 and SMN2 are deleted. We also found two novel point mutations, an A insertion at codon 8 (AGT→AAGT) and an A substitution at codon 228 (TTA→TAA).

Molecular analysis of survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes of spinal muscular atrophy patients and their parents.

We have assayed deletions of two candidate genes for spinal muscular atrophy (SMA), the survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes, in 101 patients from 86 Chinese SMA families. Deletions of exons 7 and 8 of the telomeric SMN gene were detected in 100%, 78.6%, 96.6%, and 16.7%, in type I, II, III, and adult-onset SMA patients, respectively. Deletion of exon 7 only was found in eight type II and one type III patient. One type II patient did not have a deletion of either exon 7 or 8. The prevalence of deletions of exons 5 and 6 of the NAIP gene were 22.5% and 2.4% in type I and II SMA patients, respectively. We also examined four polymorphisms of SMN genes and found that there were only two, SMN-2 and CBCD541-2, in Chinese subjects. In our study, analysis of the ratio of the telomeric to centromeric portion (T/C ratio) of the SMN gene after enzyme digestion was performed to differentiate carriers, normals, and SMA patients. We found the T/C ratio of exon 7 of the SMN gene differed significantly among the three groups, and may be used for carrier analysis. An asymptomatic individual with homozygous deletion of exons 7 and 8 of the SMN gene showed no difference in microsatellite markers in the SMA-related 5q11.2-5q13.3. In conclusion, SMN deletion in clinically presumed child-onset SMA should be considered as confirmation of the diagnosis. However, adult-onset SMA, a heterogeneous disease with phenotypical similarities to child-onset SMA, may be caused by SMN or other gene(s).