Dian Kesumapramudya Nurputra

Spinal muscular atrophy: from gene discovery to clinical trials.

Spinal muscular atrophy (SMA) is a common neuromuscular disorder with autosomal recessive inheritance, resulting in the degeneration of motor neurons. The incidence of the disease has been estimated at 1 in 6000–10,000 newborns with a carrier frequency of 1 in 40–60. SMA is caused by mutations of the SMN1 gene, located on chromosome 5q13. The gene product, survival motor neuron (SMN) plays critical roles in a variety of cellular activities. SMN2, a homologue of SMN1, is retained in all SMA patients and generates low levels of SMN, but does not compensate for the mutated SMN1. Genetic analysis demonstrates the presence of homozygous deletion of SMN1 in most patients, and allows screening of heterozygous carriers in affected families. Considering high incidence of carrier frequency in SMA, population‐wide newborn and carrier screening has been proposed. Although no effective treatment is currently available, some treatment strategies have already been developed based on the molecular pathophysiology of this disease. Current treatment strategies can be classified into three major groups: SMN2‐targeting, SMN1‐introduction, and non‐SMN targeting. Here, we provide a comprehensive and up‐to‐date review integrating advances in molecular pathophysiology and diagnostic testing with therapeutic developments for this disease including promising candidates from recent clinical trials.

Valproic acid increases SMN2 expression and modulates SF2/ASF and hnRNPA1 expression in SMA fibroblast cell lines

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is caused by loss of the survival motor neuron gene, SMN1. SMA treatment strategies have focused on production of the SMN protein from the almost identical gene, SMN2. Valproic acid (VPA) is a histone deacetylase inhibitor that can increase SMN levels in some SMA cells or SMA patients through activation of SMN2 transcription or splicing correction of SMN2 exon 7. It remains to be clarified what concentration of VPA is required and by what mechanisms the SMN production from SMN2 is elicited. We observed that in two fibroblast cell lines from Japanese SMA patients, more than 1mM of VPA increased SMN2 expression at both the transcript and protein levels. VPA increased not only full-length (FL) transcript level but also exon 7-excluding (Δ7) transcript level in the cell lines and did not change the ratio of FL/Δ7, suggesting that SMN2 transcription was mainly activated. We also found that VPA modulated splicing factor expression: VPA increased the expression of splicing factor 2/alternative splicing factor (SF2/ASF) and decreased the expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). In conclusion, more than 1mM of VPA activated SMN2 transcription and modulated the expression of splicing factors in our SMA fibroblast cell lines.

Spinal Muscular Atrophy Patient Detection and Carrier Screening Using Dried Blood Spots on Filter Paper

AIM Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder. It is caused by mutations in the SMN1, and its clinical severity is modified by copy number variations of the SMN2. According to previous studies, deletion of SMN1 exon 7 is the most frequently observed in patients with SMA. Therefore, molecular analyses exploiting this genetic lesion could be beneficial in the diagnosis of SMA. Unfortunately, in many geographical regions, physicians do not have the latest molecular screening technologies at their immediate disposal. Thus, to overcome this issue, we developed an SMA-diagnosing system using dried blood spots (DBS) placed on filter paper to facilitate remote diagnosis. METHODS In this study, we validate the applicability of DBS on Flinders Technology Associates (FTA) filter paper for detecting SMN1 exon 7 deletions and copy number variations of SMN1 and SMN2. To detect exon 7 deletions in SMN1, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis was conducted by using DNA extracted from the DBS on FTA filter paper that had been stored at room temperature for a period of up to 4 years. To determine the copy numbers of SMN1 and SMN2, we carried out SYBR green-based real-time PCR by using the same blood specimens. RESULTS The results obtained from the DBS on FTA filter paper were in complete concordance with those analyses using fresh blood specimens. This indicates that DBS on filter papers is a reliable method for SMA patient detection and carrier screenings. CONCLUSION The SMA-diagnosing system, combined with the mailing of DBS on filter paper, will be beneficial for patients suffering from neuromuscular disorders in areas with limited or no access to diagnostic facilities with molecular capabilities.

Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is currently incurable. SMA is caused by decreased levels of the survival motor neuron protein (SMN), as a result of loss or mutation of SMN1. Although the SMN1 homolog SMN2 also produces some SMN protein, it does not fully compensate for the loss or dysfunction of SMN1. Salbutamol, a β2-adrenergic receptor agonist and well-known bronchodilator used in asthma patients, has recently been shown to ameliorate symptoms in SMA patients. However, the precise mechanism of salbutamol action is unclear. We treated SMA fibroblast cells lacking SMN1 and HeLa cells with salbutamol and analyzed SMN2 mRNA and SMN protein levels in SMA fibroblasts, and changes in SMN protein ubiquitination in HeLa cells. Salbutamol increased SMN protein levels in a dose-dependent manner in SMA fibroblast cells lacking SMN1, though no significant changes in SMN2 mRNA levels were observed. Notably, the salbutamol-induced increase in SMN was blocked by a protein kinase A (PKA) inhibitor and deubiquitinase inhibitor, respectively. Co-immunoprecipitation assay using HeLa cells showed that ubiquitinated SMN levels decreased in the presence of salbutamol, suggesting that salbutamol inhibited ubiquitination. The results of this study suggest that salbutamol may increase SMN protein levels in SMA by inhibiting ubiquitin-mediated SMN degradation via activating β2-adrenergic receptor-PKA pathways.

Two Japanese Patients With SMA Type 1 Suggest that Axonal-SMN May Not Modify the Disease Severity.

BACKGROUND Spinal muscular atrophy is caused by survival motor neuron gene SMN1 mutations. SMN1 produces a full-length SMN1 protein isoform encoded by exons 1-7, and an axonal-SMN protein isoform encoded by exons 1-3 and intron 3. The axonal-SMN protein is expressed only in the embryonic period and plays a significant role in axonal growth. However, there has been no report on contribution of axonal-SMN to spinal muscular atrophy severity until now. PATIENTS Two Japanese boys with spinal muscular atrophy type 1 in our study presented with generalized muscle weakness and respiratory insufficiency soon after birth and required an artificial ventilator from early infancy. Patient 1 was compound heterozygous for two SMN1 mutations, whole-gene deletion, and an intragenic mutation (c.819_820insT). He retained one copy of SMN1 producing the N-terminal part of SMN1 including axonal-SMN. On the other hand, patient 2 was homozygous for SMN1 deletion. Both of them showed the same copy number of spinal muscular atrophy-modifying genes, NAIP and SMN2. These findings suggested that the C-terminal domain of full-length SMN1 determined the severity, irrespective of presence or absence of axonal-SMN expression. CONCLUSION In patient 1, the C-terminal domain of full-length SMN1 determined spinal muscular atrophy severity, rather than the axonal-SMN, one copy of which could be present and intact. The presence or absence of axonal-SMN may not impact disease severity in spinal muscular atrophy type 1 patients.

Paramyotonia congenita: From clinical diagnosis to in silico protein modeling analysis

Background:  Paramyotonia congenita (PMC) is an autosomal dominant disorder characterized by cold‐ or exercise‐induced myotonia. PMC is caused by a mutation in SCN4A which encodes the α‐subunit of the skeletal muscle sodium channel.

A Study of valproic acid for patients with spinal muscular atrophy

Valproic acid (VPA) is expected to become an effective therapeutic agent for spinal muscular atrophy (SMA) because of its histone deacetylase inhibitor effect.

Trinucleotide insertion in the SMN2 promoter may not be related to the clinical phenotype of SMA

BACKGROUND More than 90% of spinal muscular atrophy (SMA) patients show homozygous deletion of SMN1 (survival motor neuron 1). They retain SMN2, a highly homologous gene to SMN1, which may partially compensate for deletion of SMN1. Although the promoter sequences of these two genes are almost identical, a GCC insertion polymorphism has been identified at c.-320_-321 in the SMN1 promoter. We have also found this insertion polymorphism in an SMN2 promoter in an SMA patient (Patient A) who has SMA type 2/3. PURPOSE The aims of this study were to determine the frequency of the GCC insertion polymorphism in SMA patients, and to evaluate its effect on SMN transcription efficiency. PATIENTS AND METHODS Fifty-one SMA patients, including Patient A, were involved in this study. SMN2 transcript levels in white blood cells were measured by real-time polymerase chain reaction. Screening of the GCC insertion polymorphism was performed using denaturing high-pressure liquid chromatography. The transcription efficiency of the promoter with the insertion mutation was evaluated using a reporter-gene assay. RESULTS All SMA patients in this study were homozygous for SMN1 deletion. Patient A retained two copies of SMN2, and showed only a small amount of SMN2 transcript in white blood cells. We detected a GCC insertion polymorphism at c.-320_-321 only in Patient A, and not in 50 other SMA patients. The polymorphism had a slight but significant negative effect on transcription efficiency. DISCUSSION AND CONCLUSION Patient A was judged to be an exceptional case of SMA, because the GCC insertion polymorphism rarely exists in SMN1-deleted SMA patients. The GCC insertion polymorphism did not enhance the transcriptional efficiency of SMN2. Thus, this GCC insertion polymorphism in the SMN2 promoter may not be associated with the milder phenotype of the patient. Patient A suggests that there are other unknown factors modifying the clinical phenotype of SMA.

Molecular pathology of Sandhoff disease with p.Arg505Gln in HEXB: application of simulation analysis.

Sandhoff disease is a GM2 gangliosidosis caused by mutations in HEXB encoding the β-subunit of β-hexosaminidase A. β-Hexosaminidase A exists as a heterodimer consisting of α- and β-subunits, and requires a GM2 activator protein to hydrolyze GM2. To investigate the molecular pathology in an adult Sandhoff disease patient with an early disease onset, we performed mutation detection, western blot analysis and molecular simulation analysis. The patient had compound heterozygous mutations p.Arg505Gln and p.Ser341ValfsX30. Western blot analysis showed that the amount of mature form of the α- and β-subunits was markedly decreased in the patient. We then performed docking simulation analysis of the α- and β-subunits with p.Arg505Gln, the GM2AP/GM2 complex and β-hexosaminidase A, and GM2 and β-hexosaminidase A. Simulation analysis showed that p.Arg505Gln impaired each step of molecular conformation of the α- and β-subunits heterodimer, the activator protein and GM2. The results indicated that p.Ser341ValfsX30 reduced the amount of β-subunit, and that p.Arg505Gln hampered the maturation of α- and β-subunits, and hindered the catalytic ability of β-hexosaminidase A. In conclusion, various methods including simulation analysis were useful to understand the molecular pathology in Sandhoff disease.

Gestational Age-Dependent Increase of Survival Motor Neuron Protein in Umbilical Cord-Derived Mesenchymal Stem Cells

Background Spinal muscular atrophy (SMA) is the most common genetic neurological disease leading to infant death. It is caused by loss of survival motor neuron (SMN) 1 gene and subsequent reduction of SMN protein in motor neurons. Because SMN is ubiquitously expressed and functionally linked to general RNA metabolism pathway, fibroblasts (FBs) are most widely used for the assessment of SMN expression in SMA patients but usually isolated from skin biopsy samples after the onset of overt symptoms. Although recent translational studies of SMN-targeted therapies have revealed the very limited time window for effective SMA therapies during perinatal period, the exact time point when SMN shortage became evident is unknown in human samples. In this study, we analyzed SMN mRNA and protein expression during perinatal period by using umbilical cord-derived mesenchymal stem cells (UC-MSCs) obtained from preterm and term infants. Methods UC-MSCs were isolated from 16 control infants delivered at 22–40 weeks of gestation and SMA fetus aborted at 19 weeks of gestation (UC-MSC-Control and UC-MSC-SMA). FBs were isolated from control volunteer and SMA patient (FB-Control and FB-SMA). SMN mRNA and protein expression in UC-MSCs and FBs was determined by RT-qPCR and Western blot. Results UC-MSC-Control and UC-MSC-SMA expressed the comparable level of MSC markers on their cell surface and were able to differentiate into adipocytes, osteocytes, and chondrocytes. At steady state, SMN mRNA and protein expression was decreased in UC-MSC-SMA compared to UC-MSC-Control, as observed in FB-SMA and FB-Control. In response to histone deacetylase inhibitor valproic acid, SMN mRNA and protein expression in UC-MSC-SMA and FB-SMA was increased. During perinatal development from 22 to 40 weeks of gestation, SMN mRNA and protein expression in UC-MSC-Control was positively correlated with gestational age. Conclusion UC-MSCs isolated from 17 fetus/infant of 19–40 weeks of gestation are expressed functional SMN mRNA and protein. SMN mRNA and protein expression in UC-MSCs is increased with gestational age during perinatal development.