Multidisciplinary approach on divergent outcomes in spinal muscular atrophies: comparing DYNC1H1 and SMN1 gene mutations.

Spinal Muscular Atrophy (SMA) emerges as a prominent genetic neuromuscular disorder primarily caused by variants in the survival motor neuron (SMN) gene. However, it is noteworthy that alternative variants impacting DYNC1H1 have also been linked to a subtype known as spinal muscular atrophy lower extremity predominant (SMA-LED). This observation underscores the complexity of SMA and highlights the necessity for tailored, gene-specific management strategies. Our study elucidates how similar approaches to managing SMA can yield distinct outcomes, emphasizing the imperative for personalized gene-based interventions in effectively addressing these conditions. Two patients were referred for further management due to clinical suspicion of type-3 SMA. The definitive diagnosis was confirmed through the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique, as well as whole-exome sequencing (WES). The analysis revealed deletions in exon-7 and 8 of SMN1 in the first patient and a likely pathogenic mutation (NM_001376.5(DYNC1H1):c.1867 T > C (NP_001367.2: p.Phe623Leu)) in DYNC1H1 in the second patient. Both patients presented with lower limb muscle weakness. However, while the first patient exhibited a gradual increase in severity over the years, the second patient displayed no progressive symptoms. The management was adjusted accordingly based on the genetic findings. Our observation underscores the complexity of SMA and highlights the necessity for tailored, gene-specific management strategies. Our study elucidates how similar approaches to managing SMA can yield distinct outcomes, emphasizing the imperative for personalized gene-based interventions in effectively addressing these conditions.

Association between glaucoma susceptibility with combined defects in mitochondrial oxidative phosphorylation and fatty acid beta oxidation.

Glaucoma is one of the leading causes of visual impairment and blindness worldwide, and is characterized by the progressive damage of retinal ganglion cells (RGCs) and the atrophy of the optic nerve head (ONH). The exact cause of RGC loss and optic nerve damage in glaucoma is not fully understood. The high energy demands of these cells imply a higher sensitivity to mitochondrial defects. Moreover, it has been postulated that the optic nerve is vulnerable towards damage from oxidative stress and mitochondrial dysfunction. To investigate this further, we conducted a pooled analysis of mitochondrial variants related to energy production, specifically focusing on oxidative phosphorylation (OXPHOS) and fatty acid ß-oxidation (FAO). Our findings revealed that patients carrying non-synonymous (NS) mitochondrial DNA (mtDNA) variants within the OXPHOS complexes had an almost two-fold increased risk of developing glaucoma. Regarding FAO, our results demonstrated that longer-chain acylcarnitines (AC) tended to decrease, while shorter-chain AC tended to increase in patients with glaucoma. Furthermore, we observed that the knocking down cpt1a (a key rate-limiting enzyme involved in FAO) in zebrafish induced a degenerative process in the optic nerve and RGC, which resembled the characteristics observed in glaucoma. In conclusion, our study provides evidence that genes encoding mitochondrial proteins involved in energy metabolisms, such as OXPHOS and FAO, are associated with glaucoma. These findings contribute to a better understanding of the molecular mechanisms underlying glaucoma pathogenesis and may offer potential targets for therapeutic interventions in the future.

A systematic review on Treacher Collins syndrome: Correlation between molecular genetic findings and clinical severity.

Treacher Collins syndrome (TCS, OMIM: 154500) is a rare congenital craniofacial disorder that is caused by variants in the genes TCOF1, POLR1D, POLR1C, and POLR1B. Studies on the association between phenotypic variability and their relative variants are very limited. This systematic review summarized the 53 literatures from PubMed and Scopus to explore the potential TCS genotype-phenotype correlations with statistical analysis. Studies reporting both complete molecular genetics and clinical data were included. We identified that the molecular anomaly within TCOF1 (88.71%) accounted for most TCS cases. The only true hot spot for TCOF1 was detected in exon 24, with recurrent c.4369_4373delAAGAA variant is identified. While the hot spot for POLR1D, POLR1C, and POLR1B were identified in exons 3, 8, and 15, respectively. Our result suggested that the higher severity level was likely to be observed in Asian patients harboring TCOF1 variants rather than POLR1. Moreover, common 5-bp deletions tended to have a higher severity degree in comparison to any variants within exon 24 of TCOF1. In summary, this report suggested the relationship between genetic and clinical data in TCS. Our findings could be used as a reference for clinical diagnosis and further biological studies.

Clinical characterizations of three adults with genetically confirmed spinal muscular atrophy: a case series.

BACKGROUND: Spinal muscular atrophy is a recessively inherited autosomal neuromuscular disorder, with characteristic progressive muscle weakness. Most spinal muscular atrophy cases clinically manifest during infancy or childhood, although it may first manifest in adulthood. Although spinal muscular atrophy has come to the era of newborn screening and promising treatments, genetically confirmed spinal muscular atrophy patients are still rare in third world countries, including Indonesia. CASE PRESENTATIONS: We presented three Indonesian patients with spinal muscular atrophy genetically confirmed during adulthood. The first case was a 40-year-old male who presented with weakness in his lower limbs that started when he was 9 years old. At the age of 16 years, he could no longer walk and started using a wheelchair. He first came to our clinic at the age of 38 years, and was diagnosed with spinal muscular atrophy 2 years later. The second patient was a 58-year-old male who presented with lower limb weakness since he was 12 years old. Owing to the geographical distance and financial problems, he was referred to our clinic at the age of 56 years, when he already used a walker to walk. Lastly, the third patient was a 28-year-old woman, who was in the first semester of her second pregnancy, and who presented with slowly progressing lower limb weakness. Her limb weakness began at the age of 8 years, and slowly progressed until she became dependent on her wheelchair 8 years later until now. She had successfully given birth to a healthy daughter 3 years before her first visit to our clinic. All three patients were diagnosed with neuromuscular disorder diseases, with the differential diagnoses of Duchenne muscular dystrophy, spinal muscular atrophy, and Becker muscular dystrophy. These patients were finally confirmed to have spinal muscular atrophy due to SMN1 deletion by polymerase chain reaction and restriction fragment length polymorphism. CONCLUSIONS: Many genetic diseases are often neglected in developing countries owing to the difficulty in diagnosis and unavailable treatment. Our case series focused on the disease courses, diagnosis difficulties, and clinical presentations of three patients that finally lead to diagnoses of spinal muscular atrophy.

A Brief Analysis on Clinical Severity of Mandibulofacial Dysostosis Guion-Almeida Type.

OBJECTIVE: Genetic variants in EFTUD2 were proven to influence variable phenotypic expressivity in mandibulofacial dysostosis Guion-Almeida type (MFDGA) or mandibulofacial dysostosis with microcephaly (MFDM). Yet, the association between the severity of clinical findings with variants within the EFTUD2 gene has not been established. Thus, we aim to elucidate a possible genotype-phenotype correlation in MFDM. METHODS: Forty articles comprising 156 patients were evaluated. The genotype-phenotype correlation was analyzed using a chi-square or Fisher's exact test. RESULTS: The proportion of patients with MFDM was higher in Caucasian relative to Asian populations. Although, in general, there was no apparent genotype-phenotype correlation in patients with MFDM, Asians tended to have more severe clinical manifestations than Caucasians. In addition, cardiac abnormality presented in patients with intronic variants located in canonical splice sites was a predisposing factor in affecting MFDM severity. CONCLUSION: Altogether, this article provides the pathogenic variants observed in EFTUD2 and possible genotype-phenotype relationships in this disease.

Fast and noninvasive electronic nose for sniffing out COVID-19 based on exhaled breath-print recognition.

The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approach has been widely used to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, instead of using it alone, clinicians often prefer to diagnose the coronavirus disease 2019 (COVID-19) by utilizing a combination of clinical signs and symptoms, laboratory test, imaging measurement (e.g., chest computed tomography scan), and multivariable clinical prediction models, including the electronic nose. Here, we report on the development and use of a low cost, noninvasive method to rapidly sniff out COVID-19 based on a portable electronic nose (GeNose C19) integrating an array of metal oxide semiconductor gas sensors, optimized feature extraction, and machine learning models. This approach was evaluated in profiling tests involving a total of 615 breath samples composed of 333 positive and 282 negative samples. The samples were obtained from 43 positive and 40 negative COVID-19 patients, respectively, and confirmed with RT-qPCR at two hospitals located in the Special Region of Yogyakarta, Indonesia. Four different machine learning algorithms (i.e., linear discriminant analysis, support vector machine, stacked multilayer perceptron, and deep neural network) were utilized to identify the top-performing pattern recognition methods and to obtain a high system detection accuracy (88-95%), sensitivity (86-94%), and specificity (88-95%) levels from the testing datasets. Our results suggest that GeNose C19 can be considered a highly potential breathalyzer for fast COVID-19 screening.

Hybrid learning method based on feature clustering and scoring for enhanced COVID-19 breath analysis by an electronic nose.

Breath pattern analysis based on an electronic nose (e-nose), which is a noninvasive, fast, and low-cost method, has been continuously used for detecting human diseases, including the coronavirus disease 2019 (COVID-19). Nevertheless, having big data with several available features is not always beneficial because only a few of them will be relevant and useful to distinguish different breath samples (i.e., positive and negative COVID-19 samples). In this study, we develop a hybrid machine learning-based algorithm combining hierarchical agglomerative clustering analysis and permutation feature importance method to improve the data analysis of a portable e-nose for COVID-19 detection (GeNose C19). Utilizing this learning approach, we can obtain an effective and optimum feature combination, enabling the reduction by half of the number of employed sensors without downgrading the classification model performance. Based on the cross-validation test results on the training data, the hybrid algorithm can result in accuracy, sensitivity, and specificity values of (86 ± 3)%, (88 ± 6)%, and (84 ± 6)%, respectively. Meanwhile, for the testing data, a value of 87% is obtained for all the three metrics. These results exhibit the feasibility of using this hybrid filter-wrapper feature-selection method to pave the way for optimizing the GeNose C19 performance.

Managing pregnancy in a spinal muscular atrophy type III patient in Indonesia: a case report.

BACKGROUND: Spinal muscular atrophy is a genetic disorder characterized by degeneration of lower motor neurons, leading to progressive muscular atrophy and even paralysis. Spinal muscular atrophy usually associated with a defect of the survival motor neuron 1 (SMN-1) gene. Classification of spinal muscular atrophy is based on the age of onset and maximum motor function milestone achieved. Although spinal muscular atrophy can be screened for in newborns, and even confirmed earlier genetically, this remains difficult in Third World countries such as Indonesia. CASE PRESENTATION: A 28-year-old Asian woman in the first trimester of her second pregnancy, was referred to the neurology department from the obstetric department. Her milestone history showed she was developmentally delayed and the ability to walk independently was reached at 26 months old. At 8 years old, she started to stumble and lose balance while walking. At this age, spinal muscular atrophy was suspected because of her clinical presentations, without any molecular genetic testing. She was married at the age of 25 years and was soon pregnant with her first child. At the gestational age of 32 weeks, her first pregnancy was ended by an emergency caesarean section because of premature rupture of the membranes. In this second pregnancy, she was referred early to the general hospital from the district hospital to receive multidisciplinary care. She and her first daughter underwent genetic testing for spinal muscular atrophy, which has been readily available in our institution since 2018, to confirm the diagnosis and prepare for genetic counseling. CONCLUSIONS: Managing pregnancy in a patient with spinal muscular atrophy should be performed collaboratively. In this case, genetic testing of spinal muscular atrophy and the collaborative management of this patient allowed the clinical decision making and genetic counseling throughout her pregnancy and delivery.

Dried Blood Spot Screening System for Spinal Muscular Atrophy with Allele-Specific Polymerase Chain Reaction and Melting Peak Analysis.

Background and Aim: Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance caused by homozygous SMN1 deletions. Although SMA has been considered as incurable, newly developed drugs improve life prognoses and motor functions of patients. To maximize the efficacy of the drugs, SMA patients should be treated before symptoms become apparent. Thus, newborn screening for SMA is strongly recommended. In this study, we aim to establish a new simple screening system based on DNA melting peak analysis. Materials and Methods: A total of 124 dried blood spot (DBS) on FTA® ELUTE cards (51 SMN1-deleted patients with SMA, 20 carriers, and 53 controls) were punched and subjected to direct amplification of SMN1 and CFTR (reference gene). Melting peak analyses were performed to detect SMN1 deletions from DBS samples. Results: A combination of allele-specific polymerase chain reaction (PCR) and melting peak analyses clearly distinguished the DBS samples with and without SMN1. Compared with the results of fresh blood samples, our new system yielded 100% sensitivity and specificity. The advantages of our system include (1) biosafe collection, transfer, and storage for DBS samples, (2) obviating the need for DNA extraction from DBS preventing contamination, (3) preclusion of fluorescent probes leading to low PCR cost, and (4) fast and high-throughput screening for SMN1 deletions. Conclusion: We demonstrate that our system would be applicable to a real-world newborn screening program for SMA, because our new technology is efficient for use in routine clinical laboratories that do not have highly advanced PCR instruments.

Assessment of Spinal Muscular Atrophy Carrier Status by Determining SMN1 Copy Number Using Dried Blood Spots.

Spinal muscular atrophy (SMA) is a common neuromuscular disease with autosomal recessive inheritance. The disease gene, SMN1, is homozygously deleted in 95% of SMA patients. Although SMA has been an incurable disease, treatment in infancy with newly developed drugs has dramatically improved the disease severity. Thus, there is a strong rationale for newborn and carrier screening for SMA, although implementing SMA carrier screening in the general population is controversial. We previously developed a simple, accurate newborn SMA screening system to detect homozygous SMN1 deletions using dried blood spots (DBS) on filter paper. Here, we modified our previous system to detect the heterozygous deletions of SMN1, which indicates SMA carrier status. The system involves a calibrator-normalized relative quantification method using quantitative nested PCR technology. Our system clearly separated the DBS samples with one SMN1 copy (carrier status with a heterozygous deletion of SMN1) from the DBS samples with two SMN1 copies (non-carrier status with no deletion of SMN1). We also analyzed DBS samples from SMA families, confirmed SMA in the affected children, and determined the carrier status of their parents based on the SMN1 copy number. In conclusion, our system will provide essential information for risk assessment and genetic counseling, at least for SMA families.

Determining the association between polymorphisms of the DAT1 and DRD4 genes with attention deficit hyperactivity disorder in children from Java Island.

Attention deficit hyperactivity disorder (ADHD) is one of the most common neurobehavioural in the children. Genetic factor is known one of the factors which contributed in ADHD development. VNTR polymorphism in 3'UTR exon 15 of DAT1 gene and exon 3 of DRD4 gene are reported to be associated in ADHD. In this study we examine the association of ADHD with VNTR polymorphism of DAT1 and DRD4 gene in Indonesian children. Sixty-five ADHD children and 70 normal children (6-13 years of age), were included in the study, we matched by age and gender. ADHD was diagnosed by DSM-IV. We performed a casecontrol study to found the association between ADHD and VNTR polymorphism of DAT1 and DRD4 genes. The 10-repeat allele of DAT1 and 2-repeat allele of DRD4 were higher in Indonesian children. Although the frequency of these allele was higher, but it was similar both in ADHD and control groups. Neither DAT1 nor DRD4 gene showed showed significant difference in genotype distribution and frequency allele between both groups (p > 0.05). No association between ADHD and VNTR polymorphism of DAT1 and DRD4 genes found in Indonesian children. This data suggest that DAT1 and DRD4 do not contribute to etiology of ADHD in Indonesian children. Further studies are needed to clarify association between VNTR polymorphism of DAT1 and DRD4 genetic with ADHD of Indonesian children in larger sample size and family based study.

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.

A Rapid, Accurate and Simple Screening Method for Spinal Muscular Atrophy: High-Resolution Melting Analysis Using Dried Blood Spots on Filter Paper.

BACKGROUND: Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutation of the survival of the motor neuron 1 (SMN1) gene. More than 95% of SMA patients carry a homozygous deletion of SMN1. SMA can be screened for by polymerase chain reaction and high-resolution melting analysis (PCR-HRMA) using DNA extracted from dried blood spots (DBSs) stored on filter paper. However, there are two major problems with this approach. One is the frequent poor quality/quantity of DNA extracted from DBSs on filter paper, and the other is the difficulty in designing primer sets or probes to separate allele-specific melting curves. In this study, we addressed these problems and established a rapid, accurate and simple screening system for SMA with PCR-HRMA using DNA extracted from DBSs on filter paper. METHODS: Seventy individuals were assayed in this study, 42 SMA patients and 28 controls, all of whom had been previously been screened for SMA by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) using DNA extracted from freshly collected blood. In this study, the DNA of each individual was extracted from dried blood that had been spotted onto cards and stored at room temperature (20 - 25 degrees C) for between 1 and 8 years. PCR amplification of 30 or 45 cycles was performed using 50 ng of DNA and was immediately followed by HRMA. SMN1 and SMN2 products were co-amplified using a previously designed primer set (R111 and 541C770) containing two single nucleotide differences. RESULTS: The absorbance ratio at 260/280 of DNA extracted from DBSs ranged from 1.49 to 2.1 (mean ± SD; 1.66 ± 0.12), suggesting high-purity DNA. Thirty cycles of PCR amplification were insufficient to amplify the target alleles; PCR with 45 cycles was, however, successful in 69 out of 70 samples. PCR-HRMA using the R111/541C770 primer set enabled separation of the normalized melting curves of the samples with no SMN1 from those with SMN1 and SMN2. CONCLUSIONS: DBSs on filter paper can be a good source of DNA for the diagnosis of diseases and PCR-HRMA using DNA extracted from DBSs is an alternative method to detect the SMN1 deletion. These findings suggest that the SMA screening system using PCR-HRMA with DBSs on filter paper is practicable in a large population study over a long time period.

Attention Deficit/Hyperactivity Disorder (ADHD): age related change of completion time and error rates of Stroop test.

BACKGROUND: Attention Deficit/Hyperactivity Disorder (ADHD) is a common neurobehavioral problem in children throughout the world. The Stroop test has been widely used for the evaluation of ADHD symptoms. However, the age-related change of the Stroop test results has not been fully clarified until now. METHODS: Sixty-five ADHD and 70 age-matched control children aged 6-13 years were enrolled in this study. ADHD was diagnosed based on DSM-IV criteria. We examined the completion time and error rates of the Congruent Stroop test (CST) and Incongruent Stroop test (IST) in ADHD and control children. RESULTS: No significant difference was observed in the completion time for CST or IST between the ADHD and control children at 6-9 years old. However, ADHD children at 10-13 years old showed significantly delayed completion time for the CST and IST compared with controls of the same age. As for the error rates of the CST and IST, ADHD and control children at 6-9 years old showed no difference. However, error rates of CST and IST in the ADHD children at 10-13 years were significantly higher than those of control of the same age. CONCLUSIONS: Age may influence the results of Stroop test in ADHD children. For the ages of 10-13 years old, the Stroop test clearly separates ADHD children from control children, suggesting that it may be a useful screening tool for ADHD among preadolescent children.

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.

SMA screening system using dried blood spots on filter paper: application of COP-PCR to the SMN1 deletion test.

BACKGROUND: Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletions. Thus, the SMN1 deletion test should be performed initially as part of the diagnostic process. However, SMN2, a highly homologous gene, hampers detection of SMN1 deletion. To differentiate between SMN1 and SMN2, many analysis methods have been developed yet they are not all available worldwide. AIM: To establish a simple but accurate SMN1-deletion detection system that can be used worldwide. METHODS: Fifty DNA samples (29 SMA patients and 21 controls) from dried blood spots (DBS) on filter paper were assayed. All participants had previously been screened for SMA by PCR-restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from freshly collected blood. DNA was extracted from DBS that had been stored at room temperature (20-25℃) for between 1 and 8 years. Competitive oligonucleotide priming-PCR (COP-PCR) was performed to distinguish SMN1 and SMN2 exon7. RESULTS: DNA yield from an 11-mm diameter DBS circle was 21,171 ± 7,485 ng (mean ± SD), with an 260/280 OD ratio from 1.49 to 2.1(mean ± SD; 1.67 ±0.13). Nucleotide sequencing confirmed gene-specific amplification of SMN1 and SMN2 by COP-PCR. SMN1 and SMN2 COP-PCR results are completely consistent with those obtained by PCR-RFLP. CONCLUSION: We have combined DNA extraction from DBS on filter paper with COP-PCR that specifically detects SMN1 and SMN2, establishing a new SMN1-deletion detection system with practical application worldwide.

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 ß2-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 ß2-adrenergic receptor-PKA pathways.

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.

Intragenic mutations in SMN1 may contribute more significantly to clinical severity than SMN2 copy numbers in some spinal muscular atrophy (SMA) patients.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by deletion or intragenic mutation of SMN1. SMA is classified into several subtypes based on clinical severity. It has been reported that the copy number of SMN2, a highly homologous gene to SMN1, is associated with clinical severity among SMA patients with homozygous deletion of SMN1. The purpose of this study was to clarify the genotype-phenotype relationship among the patients without homozygous deletion of SMN1. METHODS: We performed molecular genetic analyses of SMN1 and SMN2 in 112 Japanese patients diagnosed as having SMA based on the clinical findings. For the patients retaining SMN1, the PCR or RT-PCR products of SMN1 were sequenced to identify the mutation. RESULTS: Out of the 112 patients, 106 patients were homozygous for deletion of SMN1, and six patients were compound heterozygous for deletion of one SMN1 allele and intragenic mutation in the retained SMN1 allele. Four intragenic mutations were identified in the six patients: p.Ala2Val, p.Trp92Ser, p.Thr274TyrfsX32 and p.Tyr277Cys. To the best of our knowledge, all mutations except p.Trp92Ser were novel mutations which had never been previously reported. According to our observation, clinical severity of the six patients was determined by the type and location of the mutation rather than SMN2 copy number. CONCLUSION: SMN2 copy number is not always associated with clinical severity of SMA patients, especially SMA patients retaining one SMN1 allele.