Genetic and expression studies of SMN2 gene in Russian patients with spinal muscular atrophy type II and III

<p>Abstract</p> <p>Background</p> <p>Spinal muscular atrophy (SMA type I, II and III) is an autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron gene (<it>SMN1</it>). <it>SMN2 </it>is a centromeric copy gene that has been characterized as a major modifier of SMA severity. SMA type I patients have one or two <it>SMN2 </it>copies while most SMA type II patients carry three <it>SMN2 </it>copies and SMA III patients have three or four <it>SMN2 </it>copies. The <it>SMN1 </it>gene produces a full-length transcript (FL-SMN) while <it>SMN2 </it>is only able to produce a small portion of the FL-SMN because of a splice mutation which results in the production of abnormal SMNΔ7 mRNA.</p> <p>Methods</p> <p>In this study we performed quantification of the <it>SMN2 </it>gene copy number in Russian patients affected by SMA type II and III (42 and 19 patients, respectively) by means of real-time PCR. Moreover, we present two families consisting of asymptomatic carriers of a homozygous absence of the <it>SMN1 </it>gene. We also developed a novel RT-qPCR-based assay to determine the FL-SMN/SMNΔ7 mRNA ratio as SMA biomarker.</p> <p>Results</p> <p>Comparison of the <it>SMN2 </it>copy number and clinical features revealed a significant correlation between mild clinical phenotype (SMA type III) and presence of four copies of the <it>SMN2 </it>gene. In both asymptomatic cases we found an increased number of <it>SMN2 </it>copies in the healthy carriers and a biallelic <it>SMN1 </it>absence. Furthermore, the novel assay revealed a difference between SMA patients and healthy controls.</p> <p>Conclusions</p> <p>We suggest that the <it>SMN2 </it>gene copy quantification in SMA patients could be used as a prognostic tool for discrimination between the SMA type II and SMA type III diagnoses, whereas the FL-SMN/SMNΔ7 mRNA ratio could be a useful biomarker for detecting changes during SMA pharmacotherapy.</p

Decay in survival motor neuron and plastin 3 levels during differentiation of iPSC-derived human motor neurons

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in Survival Motor Neuron 1 (SMN1), leading to degeneration of alpha motor neurons (MNs) but also affecting other cell types. Induced pluripotent stem cell (iPSC)-derived human MN models from severe SMA patients have shown relevant phenotypes. We have produced and fully characterized iPSCs from members of a discordant consanguineous family with chronic SMA. We differentiated the iPSC clones into ISL-1+/ChAT+ MNs and performed a comparative study during the differentiation process, observing significant differences in neurite length and number between family members. Analyses of samples from wild-type, severe SMA type I and the type IIIa/IV family showed a progressive decay in SMN protein levels during iPSC-MN differentiation, recapitulating previous observations in developmental studies. PLS3 underwent parallel reductions at both the transcriptional and translational levels. The underlying, progressive developmental decay in SMN and PLS3 levels may lead to the increased vulnerability of MNs in SMA disease. Measurements of SMN and PLS3 transcript and protein levels in iPSC-derived MNs show limited value as SMA biomarkers

Revised Hammersmith Scale for Spinal Muscular Atrophy: : A SMA specific clinical outcome assessment tool

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Recent translational research developments in Spinal Muscular Atrophy (SMA), outcome measure design and demands from regulatory authorities require that clinical outcome assessments are 'fit for purpose'. An international collaboration (SMA REACH UK, Italian SMA Network and PNCRN USA) undertook an iterative process to address discontinuity in the recorded performance of the Hammersmith Functional Motor Scale Expanded and developed a revised functional scale using Rasch analysis, traditional psychometric techniques and the application of clinical sensibility via expert panels. Specifically, we intended to develop a psychometrically and clinically robust functional clinician rated outcome measure to assess physical abilities in weak SMA type 2 through to strong ambulant SMA type 3 patients. The final scale, the Revised Hammersmith Scale (RHS) for SMA, consisting of 36 items and two timed tests, was piloted in 138 patients with type 2 and 3 SMA in an observational cross-sectional multi-centre study across the three national networks. Rasch analysis demonstrated very good fit of all 36 items to the construct of motor performance, good reliability with a high Person Separation Index PSI 0.98, logical and hierarchical scoring in 27/36 items and excellent targeting with minimal ceiling. The RHS differentiated between clinically different groups: SMA type, World Health Organisation (WHO) categories, ambulatory status, and SMA type combined with ambulatory status (all p < 0.001). Construct and concurrent validity was also confirmed with a strong significant positive correlation with the WHO motor milestones rs = 0.860, p < 0.001. We conclude that the RHS is a psychometrically sound and versatile clinical outcome assessment to test the broad range of physical abilities of patients with type 2 and 3 SMA. Further longitudinal testing of the scale with regards change in scores over 6 and 12 months are required prior to its adoption in clinical trials.Peer reviewedFinal Published versio

Molecular Determinants of Survival Motor Neuron (SMN) Protein Cleavage by the Calcium-Activated Protease, Calpain

Spinal muscular atrophy (SMA) is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN) protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs). It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V), reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S), abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294) resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN

Validation of a high resolution NGS method for detecting spinal muscular atrophy carriers among phase 3 participants in the 1000 Genomes Project

BACKGROUND: Spinal muscular atrophy (SMA) is the most common pan-ethnic cause of early childhood death due to mutations in a single gene, SMN1. Most chromosome 5 homologs have a functional gene and dysfunctional copy, SMN2, with a single synonymous base substitution that results in faulty RNA splicing. However, the copy number of SMN1 and SMN2 is highly variable, and one in 60 adults worldwide are SMA carriers. Although population-wide screening is recommended, current SMA carrier tests have not been incorporated into targeted gene panels. METHODS: Here we describe a novel computational protocol for determining SMA carrier status based solely on individual exome data. Our method utilizes a Bayesian hierarchical model to quantify an individual’s carrier probability given only his or her SMN1 and SMN2 reads at six loci of interest. RESULTS: We find complete concordance with results obtained with the current qPCR-based testing standard in known SMA carriers and affecteds. We applied our protocol to the phase 3 cohort of the 1,000 Genomes Project and found carrier frequencies in multiple populations consistent with the present literature. CONCLUSION: Our process is a convenient, robust alternative to qPCR, which can easily be integrated into the analysis of large multi-gene NGS carrier screens. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12881-015-0246-2) contains supplementary material, which is available to authorized users

Parametric Oscillations of a Thermal Field During Explosive Crystallization of Amorphous Films

Изучены тепловые процессы, происходящие при взрывной кристаллизации аморфных пленок, напыленных на подложку. Представлены результаты численного моделирования параметрических колебаний теплового поля в системе «фазовая граница – подложка». Рассмотрены стационарный и волновой режимы возбуждения горячих центров кристаллизации в аморфной фазе. Расчеты выполнены для аморфной пленки германия. Установлены основные физические факторы, определяющие амплитудно-частотные свойства данного процесса: толщина пленки, температура подложки и скорость фазовой границы кристаллизации. Указаны примеры возникновения резонансных ситуаций. Рассмотрены колебания параметрической системы, которая испытывает внешнее воздействие в режиме биений. Для этой системы по-строен трехмерный фазовый портрет, демонстрирующий ее динамические свойства. При наличии спектра частот структура фазовых траекторий в основном аналогична варианту колебаний на одной частоте.The thermal processes occurring during explosive crystallization of amorphous films deposited on a substrate are studied. The results of numerical modeling of parametric oscillations of a thermal field in the “phase boundary – substrate” system are presented. The stationary and wave modes of hot crystallization centers in the amorphous phase are considered. The calculations are performed for an amorphous germanium film. The main physical factors determining the amplitude and frequency properties of this process are established: film thickness, substrate temperature, and crystallization phase boundary rate. Examples of the resonant situation occurrence are indicated. The parametric system oscillations, which has external influence in the beating mode, are considered. A three-dimensional phase portrait is constructed for this system, demonstrating its dynamic properties. In the presence of a frequency spectrum, the structure of phase trace is basically similar to the variant of oscillations at one frequency