Spinal muscular atrophy carriers with two SMN1 copies

Background

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder. Over 95% of SMA patients have homozygous deletions of the SMA-causative gene, SMN1. Thus, SMA carriers are usually diagnosed based on SMN1 copy number, with one copy indicating SMA carrier status. However, two SMN1 copies do not always exclude carrier status. In this study, we identified SMA carriers with two SMN1 copies.

Phenotypes of SMA patients retaining SMN1 with intragenic mutation

BackgroundSpinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous deletion or intragenic mutation of the SMN1 gene. It is well-known that high copy number of its homologous gene, SMN2, modifies the phenotype of SMN1-deleted patients. However, in the patients with intragenic SMN1 mutation, the relationship between phenotype and SMN2 copy number remains unclear.MethodsWe have analyzed a total of 515 Japanese patients with SMA-like symptoms (delayed developmental milestones, respiratory failures, muscle weakness etc.) from 1996 to 2019. SMN1 and SMN2 copy numbers were determined by quantitative polymerase chain reaction (PCR) method and/or multiplex ligation-dependent probe amplification (MLPA) method. Intragenic SMN1 mutations were identified through DNA and RNA analysis of the fresh blood samples.ResultsA total of 241 patients were diagnosed as having SMA. The majority of SMA patients showed complete loss of SMN1 (n = 228, 95%), but some patients retained SMN1 and carried an intragenic mutation in the retaining SMN1 (n = 13, 5%). Ten different mutations were identified in these 13 patients, consisting of missense, nonsense, frameshift and splicing defect-causing mutations. The ten mutations were c.275G > C (p.Trp92Ser), c.819_820insT (p.Thr274Tyrfs*32), c.830A > G (p.Tyr277Cys), c.5C > T (p.Ala2Val), c.826 T > C (p.Tyr276His), c.79C > T (p.Gln27*), c.188C > A (p.Ser63*), c.422 T > C (p.Leu141Pro), c.835-2A > G (exon 7 skipping) and c.835-3C > A (exon 7 skipping). It should be noted here that some patients with milder phenotype carried only a single SMN2 copy (n = 3), while other patients with severe phenotype carried 3 SMN2 copies (n = 4).ConclusionIntragenic mutations in SMN1 may contribute more significantly to clinical severity than SMN2 copy numbers.     

Combination of SMN2 copy number and NAIP deletion predicts disease severity in spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene. The SMN2 gene is highly homologous to SMN1 and has been reported to be correlated with severity of the disease. The clinical presentation of SMA varies from severe to mild, with three clinical subtypes (type I, type II, and type III) that are assigned according to age of onset and severity of the disease. Here, we aim to investigate the potential association between the number of copies of SMN2 and the deletion in the NAIP gene with the clinical severity of SMA in patients of Malaysian origin. Forty-two SMA patients (14 of type I, 20 type II, and 8 type III) carrying deletions of the SMN1 gene were enrolled in this study. SMN2 copy number was determined by fluorescence-based quantitative polymerase chain reaction assay. Twenty-nine percent of type I patients carried one copy of SMN2, while the remaining 71% carried two copies. Among the type II and type III SMA patients, 29% of cases carried two copies of the gene, while 71% carried three or four copies of SMN2. Deletion analysis of NAIP showed that 50% of type I SMA patients had a homozygous deletion of exon 5 of this gene and that only 10% of type II SMA cases carried a homozygous deletion, while all type III patients carried intact copies of the NAIP gene. We conclude that there exists a close relationship between SMN2 copy number and SMA disease severity, suggesting that the determination of SMN2 copy number may be a good predictor of SMA disease type. Furthermore, NAIP gene deletion was found to be associated with SMA severity. In conclusion, combining the analysis of deletion of NAIP with the assessment of SMN2 copy number increases the value of this tool in predicting the severity of SMA.     

The ultrastructure of peripheral nerve, motor end-plate and skeletal muscle in patients suffering from spinal muscular atrophy with respiratory distress type 1 (SMARD1)

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is genetically and clinically distinct from classic spinal muscular atrophy (SMA1). It results from mutations in the gene encoding immunoglobulin -binding protein 2 (IGHMBP2) on chromosome 11q13. Patients develop distally pronounced muscular weakness and early involvement of the diaphragm, resulting in respiratory failure. Sensory and autonomic nerves are also affected at later stages of the disease. We investigated peripheral nerves, skeletal muscles and neuromuscular junctions (NMJ) ultrastructurally in five unrelated patients and three siblings with genetically confirmed SMARD1. In mixed motor and sensory nerves we detected Wallerian degeneration and axonal atrophy similar to the ultrastructural findings described in SMA1. Isolated axonal atrophy was evident in purely sensory nerves. All investigated NMJ of patients with SMARD1 were dysmorphic and lacked a terminal axon. Moreover, we also observed characteristics of neuropathies, such as abnormalities in myelination, that have not been described in spinal muscular atrophies so far. Based on these findings we conclude that impairment of IGHMBP2 function leads to axonal degeneration, abnormal myelin formation, and motor end-plate degeneration.     

Correlation between SMN2 copy number and clinical phenotype of spinal muscular atrophy: three SMN2 copies fail to rescue some patients from the disease severity

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is characterized by degeneration of the anterior horn cells of the spinal cord, which leads to the axial and limb weakness associated with muscle atrophy. SMA is classified into three groups based on the clinical severity: type I (severe), type II (intermediate) and type III (mild). All three clinical subtypes of SMA are caused by mutations of the SMN1 gene. More than 95 % of SMA patients show homozygous deletion of SMN1. It is thought that SMN2, which is a highly homologous gene of SMN1, compensates for the SMN1 deletion to some degree. To clarify the relationship between SMN2 and the disease severity of SMA, we performed fluorescence-based quantitative polymerase chain reaction assay of the copy number of SMN2 in 27 patients (11 type I and 16 type II–III) homozygous for SMN1 deletion. The SMN2 copy number in type II–III patients was 3.1 ± 0.3 (mean ± SD), which is significantly higher than that observed in type I patients, 2.2 ± 0.6 (P < 0.01). However, three of the 11 type I patients carried 3 SMN2 copies. A type I patient with 3 SMN2 copies was studied further. RT-PCR analysis of the patient showed a trace of full-length SMN2 mRNA species, but a large amount of the truncated SMN2 mRNA species lacking exon 7. In conclusion, SMN2 alleles are not functionally equivalent among SMA patients, although in general the SMN2 copy number is correlated with the severity of SMA. Genetic background influencing splicing mechanisms of the SMN2 gene may be more critical in some SMA patients. Received: 11 December 2001, Received in revised form: 14 March 2002, Accepted: 19 March 2002     

The influence of storage parameters on measurement of survival motor neuron (SMN) protein levels: Implications for pre-clinical studies and clinical trials for spinal muscular atrophy

Spinal muscular atrophy (SMA) is caused by low levels of survival motor neuron (SMN) protein. A growing number of potential therapeutic strategies for SMA are entering pre-clinical and clinical testing, including gene therapy and antisense oligonucleotide-based approaches. For many such studies SMN protein levels are used as one major readout of treatment efficacy, often necessitating comparisons between samples obtained at different times and/or using different protocols. Whether differences in tissue sampling strategies or storage parameters have an influence on measurable SMN levels remains to be determined. We assessed murine SMN protein immunoreactivity over time and under differing tissue storage conditions. SMN protein levels, measured using sensitive quantitative fluorescent western blotting, declined rapidly over a period of several days following sample collection, especially when protein was extracted immediately and stored at −20 °C. Storage of samples at lower temperatures (−80 °C), and as intact tissue, led to significantly better preservation of SMN immunoreactivity. However, considerable deterioration in measurable SMN levels occurred, even under optimal storage conditions. These issues need to be taken into consideration when designing and interpreting pre-clinical and clinical SMA studies where SMN protein levels are being measured.     

Quantitative analysis of SMN2 based on real-time PCR: correlation of clinical severity and SMN2 gene dosage

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder, caused by homozygous deletion of the survival motor neuron gene 1 (SMN1). SMN2, a gene highly homologous to SMN1, is considered to influence the severity of SMA. Patients with SMA have been classified into three types on the basis of age at onset and clinical severity. In the present study, we performed a quantitative analysis of SMN2 in 22 patients of SMA to further clarify the correlation between clinical severity and SMN2 gene dosage. We determined the SMN2 gene copy number based on real-time PCR. In 3 Type I patients with only one SMN2 copy, the clinical phenotype was the severest. The remaining 14 Type I patients had two or three copies of the SMN2 gene, and showed a variable clinical severity. A patient with 3 copies required artificial ventilation at 2 months old. Five Type II and Type III patients had at least 4 copies of the SMN2 gene. Although the SMN2 gene dosage correlates the clinical severity, the mechanism by which SMN2 shows compensation in some Type I patients remains to be determined.     

Spinal muscular atrophy with respiratory distress syndrome (SMARD1): Case report and review of literature

Spinal muscular atrophy with respiratory distress syndrome (SMARD1) is a rare cause of early infantile respiratory failure and death. No cases have been currently described from India. Two low-birth-weight infants presented prior to 6 months of age with recurrent apnea and respiratory distress. Both required prolonged ventilation, and had distal arthrogryposis and diaphragmatic eventration. Nerve conduction study revealed motor sensory axonopathy. Genetic testing confirmed mutations in immunoglobulin mu binding protein (IGHMBP2). These two cases establish presence of SMARD1 in our population. Both infants died on discontinuation of ventilation. Antenatal diagnoses done in one pregnancy. Though rare, high index of suspicion is essential in view of poor outcome and aid antenatal counseling.     

Intrathecal administration of nusinersen in adult and adolescent patients with spinal muscular atrophy and scoliosis: Transforaminal versus conventional approach

Spinal deformities and surgical correction of scoliosis can make intrathecal delivery of nusinersen very challenging. We aim to evaluate the feasibility and safety of intrathecal administration of nusinersen either via interlaminar or transforaminal approach in a cohort of adult and adolescent patients with spinal muscular atrophy (SMA). Twelve patients were treated with nusinersen in our center under CT-guidance; after a CT scan of the lumbar column, we identified a safe virtual trajectory for the needle and defined patients to address to the transforaminal approach (seven patients) or the interlaminar approach (five patients). Out of 47 procedures, all injections but one were successful. There was one adverse event (post-lumbar puncture syndrome) in the interlaminar approach group (out of 20 procedures) and four adverse events in TFA group (out of 27 procedures) including one serious adverse event, a subarachnoid hemorrhage that required hospitalization. Transforaminal approach can be considered an effective option for nusinersen administration but potentially associated with serious complications, therefore it should be recommended in very selected patients.     

Nusinersen initiated in infants during the presymptomatic stage of spinal muscular atrophy: Interim efficacy and safety results from the Phase 2 NURTURE study

Spinal muscular atrophy (SMA) is a neurodegenerative disease associated with severe muscle atrophy and weakness in the limbs and trunk. We report interim efficacy and safety outcomes as of March 29, 2019 in 25 children with genetically diagnosed SMA who first received nusinersen in infancy while presymptomatic in the ongoing Phase 2, multisite, open-label, single-arm NURTURE trial. Fifteen children have two SMN2 copies and 10 have three SMN2 copies. At last visit, children were median (range) 34.8 [25.7–45.4] months of age and past the expected age of symptom onset for SMA Types I or II; all were alive and none required tracheostomy or permanent ventilation. Four (16%) participants with two SMN2 copies utilized respiratory support for ≥6 h/day for ≥7 consecutive days that was initiated during acute, reversible illnesses. All 25 participants achieved the ability to sit without support, 23/25 (92%) achieved walking with assistance, and 22/25 (88%) achieved walking independently. Eight infants had adverse events considered possibly related to nusinersen by the study investigators. These results, representing a median 2.9 years of follow up, emphasize the importance of proactive treatment with nusinersen immediately after establishing the genetic diagnosis of SMA in presymptomatic infants and emerging newborn screening efforts.     

Segmental distribution of muscle weakness in SMA III: implications for deterioration in muscle strength with time

We examined 26 spinal muscular atrophy type III (SMA III) patients with SMNt deletions, searching for possible segmental distribution of muscle weakness. In those with disease duration of ≤11 years, the weakest muscles were upper lumbar innervated ones in the lower extremities. In the upper extremities, early involvement of triceps muscle suggested the possibility of lower cervical (C7) onset. Electrophysiologically, weaker muscles had a more severe reduction in the recruitment pattern, particularly in the lower extremities. However, severe reduction in recruitment was sometimes also observed in clinically strong muscles. In patients with disease duration of ≥16 years and regardless of disease duration, in those with disease onset at ≤3 years of age, weakness and severe electrophysiological changes were more widespread. These findings may suggest a progression in muscle weakness with time. When compared to 12 patients with Becker muscular dystrophy (BMD), early stage SMA III with weak iliopsoas-strong gluteus maximus stood in contrast to BMD with weak gluteus maximus-strong iliopsoas.     

Clinical and mutational characteristics of spinal muscular atrophy with respiratory distress type 1 in the Netherlands

Spinal muscular atrophy with respiratory distress type 1 is an autosomal recessive disorder with early respiratory difficulties, distal muscle weakness, and contractures leading to foot deformities as the most striking clinical symptoms. Mutations of the gene encoding the immunoglobulin heavy chain μ-binding protein 2, mapped on chromosome 11q13, are the cause of the disease. We present the clinical and mutational characteristics of ten patients in the Netherlands who showed considerable clinical variability; they carried six novel mutations, including a deletion of exon 2. However, there were no clear phenotype–genotype correlations.     

Long-term efficacy of nusinersen and its evaluation in adolescent and adult patients with spinal muscular atrophy types 1 and 2

BackgroundsThe efficacy of nusinersen and its evaluation in patients with spinal muscular atrophy (SMA) has been established in clinical trials only for pediatric patients, not for adolescent and adult patients who developed SMA in infancy or early childhood. We report a long-term follow-up in adolescent and adult patients with SMA types 1 and 2.MethodsNusinersen-treated patients with SMA types 1 and 2 between 2017 and 2022 were retrospectively reviewed. We compared baseline motor function tests with those after the final treatment. Physical and occupational therapists performed Children’s Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND), Hammersmith Functional Motor Scale-Expanded (HFMSE), and Revised Upper Limb Module (RULM). The Landau and Galant reflexes were not performed in CHOP-INTEND. Meaningful improvement was defined as CHOP-INTEND, 4; HFSME, 3; and RULM, 2.ResultsSeven patients with SMA (type 1, 1; type 2, 6) with a median age of 23 (range, 12–40) years were treated with nusinersen for 3.55 (1.78–4.53) years. Improvement was detected in CHOP-INTEND (pre, 5 [0–31]; post, 21 [0–39]; difference, 5 [0–26]; p = 0.100) without significance, although not in HFMSE (pre, 0 [0–3]; post, 0 [0–5]; difference, 0 [0–2]; p = 0.346) and RULM (pre, 1 [0–20]; post, 3 [0–21]; difference, 1 [0–2]; p = 0.089). Owing to prolonged treatment intervals with the COVID-19 pandemic, RULM worsened in two patients.ConclusionNusinersen was effective in long-term follow-up. Only CHOP-INTEND showed meaningful improvement. The interval between doses of nusinersen should not be prolonged even with the COVID-19 pandemic.     

Handgrip and finger flexion strength in children: A cross-sectional assessment of age-related normative data and application as a clinical functional marker in paediatric neuromuscular disorders

BackgroundThe aim of this study was to evaluate handgrip and finger flexion strength (HGFS) as functional marker for disease progression in children with neuromuscular disorders (NMD) and present normative data in a paediatric healthy cohort.MethodsWe applied the fixed hand and finger dynamometer HFD 200 to assess HGFS under standardised, isometric and biomechanical conditions. In our cross-sectional study HGFS was analysed in n = 233 paediatric healthy controls (HC) and a cohort of n = 33 children with NMD between five and 18 years. In seven children with spinal muscular atrophy (SMA), HGFS were assessed prior to and under treatment with nusinersen over a two months period. HGFS of children with NMD was correlated with respiratory parameters, anthropometric data, hand function and motor scores.ResultsPatients with NMD exhibited a heterogenous HGFS pattern. HGFS was lower than in HC (p < 0.001). Children with SMA gained a significant increase in strength after two months of treatment (p < 0.05, r = 0.75–0.9).ConclusionHGFS is a sensitive functional marker in paediatric NMD to identify minimal changes in distal muscle strength. HGFS may evolve as a sensitive outcome measure to monitor upcoming therapeutic interventions in particular for non-ambulant patients with NMD.     

Screening of the LIX1 gene in Japanese and Malaysian patients with SMA and/or SMA-like disorder

Background: The majority of spinal muscular atrophy (SMA) patients showed homozygous deletion or other mutations of SMN1. However, the genetic etiology of a significant number of SMA patients has not been clarified. Recently, mutation in the gene underlying cat SMA, limb expression 1 (LIX1), has been reported. Similarity in clinical and pathological features of cat and human SMA may give an insight into possible similarity of the genetic etiology. Patients and methods: In this study, we screened for a mutation in LIX1 using direct DNA sequencing in our SMA and/or SMA-like patients who retained SMN1. A total of 33 patients were enrolled in this study, of which 22 were Japanese and 11 were Malaysians. All these patients possessed at least two copies of SMN1. Results: We did not identify any pathogenic mutations in the coding regions or splice sites of LIX1 in the patients. In addition, we described a polymorphism within LIX1 intron 3, c.387 + 107A > T. We found that A-allele is significantly more frequent in SMA patients compared to normal individuals. Conclusion: Molecular genetic analysis of our SMA and/or SMA-like patients suggests that LIX1 is not associated with the development of their disorders. However, the number of patients analyzed in this study was very limited, and a larger study with bigger sample size is needed to confirm this result.     

Therapeutic efficacy of acotiamide in patients with functional dyspepsia based on enhanced postprandial gastric accommodation and emptying: randomized controlled study evaluation by real-time ultrasonography

Background Improvement in subjective symptoms has been reported in functional dyspepsia (FD) patients administered with acotiamide. Improvement was confirmed in meal‐related symptoms, such as postprandial fullness, upper abdominal bloating, and early satiety. We examined the mechanism underlying the effects of acotiamide on gastric accommodation reflex (GAR) and gastroduodenal motility in FD patients. Methods Thirty‐four FD patients (mean age, 40.4 years) were examined ultrasonographically before and after 14–18 days of acotiamide (100 mg t.i.d.) or placebo administration. To assess GAR, expansion rate in cross‐sectional area of the proximal stomach was measured after every 100‐mL ingestion, using a straw, of up to 400 mL of a liquid meal (consommé soup, 13.1 kcal; 400 mL) in a supine position. Next, we measured gastric emptying rate (GER), motility index (MI, antral contractions), and reflux index (RI, duodenogastric reflux) to assess gastroduodenal motility. Patients also completed a survey based on the seven‐point Likert scale both before and after drug administration. Key Results Of the 37 cases, 19 and 18 were administered with acotiamide and placebo A respectively, significant difference was observed in GAR between the acotiamide and placebo groups (21.7%vs 4.4%) after 400 mL ingestion. GER significantly accelerated after treatment in the acotiamide group (P = 0.012), no significant differences were observed in MI and RI between the two groups. Improvement rates were 35.3 and 11.8% for the acotiamide and placebo groups. Conclusions & Inferences Acotiamide significantly enhances GAR and GER in FD patients. Acotiamide may have therapeutic potential for FD patients.