Cervical rotation, chest deformity and pelvic obliquity in patients with spinal muscular atrophy.

BACKGROUND: Musculoskeletal disorders are often observed in patients with spinal muscular atrophy (SMA). The aim of the study was to assess passive ranges of rotation in the cervical spine, chest deformity and pelvic obliquity in SMA patients, and to compare these results to the norms obtained in the group of healthy individuals. The second aim was to review these measurements and Cobb angle values for correlations in SMA patients. METHODS: The study included 74 patients with SMA and 89 healthy individuals aged 2 to 18 years. Cervical Rotation (CR), Supine Angle of Trunk Rotation (SATR) and Pelvic Obliquity (PO) tests were carried out. RESULTS: Cervical rotation ranges were significantly higher in the control group than in SMA patients (p < 0.05). Differences between cervical rotation ranges to the left and to the right were significantly larger in SMA I and SMA II groups than in healthy individuals (p = 0.000). Chest asymmetry and pelvic obliquity were bigger in SMA groups than in the control (p < 0.05). Significant correlations between cervical rotation measurements, chest deformity, pelvic obliquity and Cobb angle were found in SMA individuals, depending on the type. CONCLUSIONS: The results of the study suggest that CR, SATR and PO tests may assist in the assessment of SMA patients in addition to the radiographic evaluation of the spine. Biomechanical relationships between disorders located in various skeletal structures should be taken into account in the treatment of SMA patients. Special attention should be given to assessing postural parameters in non- sitters and sitters. Treatment of patients with SMA and associated musculoskeletal disorders requires a multi-specialist approach.

Spinal muscular atrophy - new therapies, new challenges.

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease with an autosomal recessive trait of inheritance and great variability of its clinical course - from the lethal congenital type (SMA0) to the adult-onset form (SMA4). The disease is associated with a deficiency of SMN protein, which is encoded by two genes SMN1 and SMN2. Clinical symptoms depend on mutations in the SMN1 gene. The number of copies of twin similar SMN2 gene, which produces small amounts of SMN protein, is the main phenotype modifier, which determines the clinical severity of the disease. Until recently, it was considered that spinal cord motoneurons undergo selective loss. Recent studies have shown the role of SMN protein in various cellular processes and the multisystemic character of SMA. The aim of the therapeutic strategies developed so far has been to increase the expression of SMN protein by modifying the splicing of SMN2 gene (intrathecally administered antisense oligonucleotide - nusinersen; orally available small molecules: RG7916 and LMI070 or SMN1 gene replacement therapy (AAV9-SMN). The first SMN2-directed antisense oligonucleotide (nusinersen) has demonstrated in clinical trials high efficiency, and it has now been registered. The best effects were obtained in patients who were introduced to the drug in the pre symptomatic period. Studies on other substances are ongoing. The great advances in SMA therapy and increased understanding of the pathogenesis of the disease raise hopes for changes to the natural history of the disease. Simultaneously, it increases awareness of the need to improve the standard of patient care and early diagnosis (newborn screening). Many questions (e.g. emerging phenotypes, combined therapies, systemic vs. intrathecal administration, long-term consequences, and complications of the therapy) will require further studies and observations.

The remarkable phenotypic variability of the p.Arg269HiS variant in the TRPV4 gene.

INTRODUCTION: Mutations in the TRPV4 gene are associated with neuromuscular disorders and skeletal dysplasias, which present a phenotypic overlap. METHODS: Next-generation sequencing and Sanger sequencing were used to analyze the TRPV4 gene. RESULTS: We present 2 Polish families with TRPV4-related disorder harboring the same p.Arg269His mutation. The disease phenotypic expression was extremely variable (from mild scapular winging to severe hypotonia, global weakness, inability to walk unaided, congenital contractures, scoliosis, and respiratory insufficiency), but did not suggest anticipation. The 2 most severely affected patients showed congenital distal contractures of the upper limbs and involvement of cranial nerves (manifesting as facial asymmetry and strabismus). The disease course seemed to be stable, although in later stages it caused respiratory insufficiency and progression of physical disability. DISCUSSION: The phenotypic variability observed in p.Arg269His carriers suggests that an additional modifier or a more complex pathogenic mechanism exists. Muscle Nerve 59:129-133, 2019.

Reliability of four tests to assess body posture and the range of selected movements in individuals with spinal muscular atrophy.

BACKGROUND: The majority of individuals with spinal muscular atrophy (SMA) experience progressive skeletal deformities which may affect the quality of life and mobility. To date, no studies have evaluated the reliability of tests assessing body posture and joint mobility in SMA patients. The purpose of this study was to assess the reliability of Cervical Rotation test (CR), Supine Angle of Trunk Rotation test (SATR), Hip Extension test (HE) and Pelvic Obliquity test (PO) developed to evaluate the musculoskeletal system in SMA individuals. METHODS: Thirty individuals (12 girls and 18 boys) aged 4-15 with SMA type II (n = 24) and III (n = 6) confirmed by genetic examinations were qualified for the study. The participants were examined twice by three physiotherapists on the same day. The examination included four tests, i.e. CR, SATR, HE and PO tests aimed at assessing ranges of rotation in the cervical spine, chest deformities, ranges of hip extension and pelvis position while sitting. Statistical calculations were made with the use of statistical software IBM SPSS Statistics version 20. Reliability was assessed using the Intraclass Correlation Coefficient (ICC). RESULTS: Intraobserver reliability was excellent for CR (ICC range 0.839-0.911), SATR (ICC range 0.918-0.939 - the upper part of the sternum; ICC range 0.951-0.975 - the lower part of the sternum), HE (ICC range 0.988-0,991) and PO (ICC range 0.896-0.935) tests. The interobserver ICC reached the excellent values in CR (ICC range 0.912-0.920), SATR (ICC = 0.888 - the upper part of the sternum, ICC = 0.951 - the lower part of the sternum), HE (ICC range 0.922-0.923) and PO (ICC = 0.928) tests. CONCLUSIONS: CR, SATR, HE and PO tests are reliable and may be used for examining individuals with SMA. The application of these tests provides a possibility to detect early changes in the musculoskeletal system in children and adolescents and to assess the effectiveness of the implemented pharmacotherapy and rehabilitation.

Optimised, Broad NGS Panel for Inherited Eye Diseases to Diagnose 1000 Patients in Poland.

Advances in gene therapy and genome editing give hope that new treatments will soon be available for inherited eye diseases that together affect a significant proportion of the adult population. New solutions are needed to make genetic diagnosis fast and affordable. This is the first study of such a large group of patients with inherited retinal dystrophies (IRD) and inherited optic neuropathies (ION) in the Polish population. It is based on four years of diagnostic analysis using a broad, targeted NGS approach. The results include the most common pathogenic variants, as well as 91 novel causative variants, including frameshifts in the cumbersome RPGR ORF15 region. The high frequency of the ABCA4 complex haplotype p.(Leu541Pro;Ala1038Val) was confirmed. Additionally, a deletion of exons 22-24 in USH2A, probably specific to the Polish population, was uncovered as the most frequent copy number variation. The diagnostic yield of the broad NGS panel reached 64.3% and is comparable to the results reported for genetic studies of IRD and ION performed for other populations with more extensive WES or WGS methods. A combined approach to identify genetic causes of all known diseases manifesting in the posterior eye segment appears to be the optimal choice given the currently available treatment options and advanced clinical trials.

270th ENMC International Workshop: Consensus for SMN2 genetic analysis in SMA patients 10-12 March, 2023, Hoofddorp, the Netherlands.

The 270th ENMC workshop aimed to develop a common procedure to optimize the reliability of SMN2 gene copy number determination and to reinforce collaborative networks between molecular scientists and clinicians. The workshop involved neuromuscular and clinical experts and representatives of patient advocacy groups and industry. SMN2 copy number is currently one of the main determinants for therapeutic decision in SMA patients: participants discussed the issues that laboratories may encounter in this molecular test and the cruciality of the accurate determination, due the implications as prognostic factor in symptomatic patients and in individuals identified through newborn screening programmes. At the end of the workshop, the attendees defined a set of recommendations divided into four topics: SMA molecular prognosis assessment, newborn screening for SMA, SMN2 copies and treatments, and modifiers and biomarkers. Moreover, the group draw up a series of recommendations for the companies manufacturing laboratory kits, that will help to minimize the risk of errors, regardless of the laboratories' expertise.

Cervical dystonia and no oculomotor apraxia as new manifestation of ataxia-telangiectasia-like disorder 1 - case report and review of the literature.

Ataxia-telangiectasia-like disorder 1 (ATLD1) is a rare neurodegenerative disorder associated with early onset ataxia and oculomotor apraxia. The genetic determination of ATLD1 is a mutation in the MRE11 gene (meiotic recombination 11 gene), which causes DNA-double strand break repair deficits. Clinical features of patients with ATLD1 resemble those of ataxia telangiectasia (AT), with slower progression and milder presentation. Main symptoms include progressive cerebellar ataxia, oculomotor apraxia, cellular hypersensitivity to ionizing radiations. Facial dyskinesia, dystonia, dysarthria have also been reported. Here we present a 45-year old woman with cervical and facial dystonia, dysarthria and ataxia, who turned out to be the first case of ATLD without oculomotor apraxia, and with dystonia as a main manifestation of the disease. She had presented those non-specific symptoms for years, before whole exome sequencing confirmed the diagnosis.

Newborn screening and gene therapy in SMA: Challenges related to vaccinations.

Spinal muscular atrophy (SMA) affects one in 7,500-10,000 newborns. Before the era of disease-modifying therapies, it used to be the major genetic cause of mortality in infants. Currently, there are three therapies approved for SMA, including two molecules modifying the splicing of the SMN2 gene and one gene therapy providing a healthy copy of the SMN gene with a viral vector. The best effects of any of these therapies are achieved when the treatment is administered in the presymptomatic stage of the disease, therefore newborn screening programs are being introduced in many countries. Patients identified in newborn screening might be eligible for gene therapy. However, gene therapy and the associated administration of steroids in newborns might interfere with the vaccination schedule, which includes live immunization against tuberculosis in some countries. The timing of gene therapy in patients who received live vaccinations has not yet been addressed neither in the clinical trials nor in the existing international guidelines. The Polish Vaccinology Association has developed the first recommendations for gene therapy administration in newborns who received live vaccination against tuberculosis. Their statement was implemented in the current guidelines for Polish SMA patients identified in the newborn screening program and might be helpful for medical professionals in other countries where live vaccine against tuberculosis is still in routine use in newborns.

Observation of the natural course of type 3 spinal muscular atrophy: data from the polish registry of spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is one of the most frequent and severe genetic diseases leading to premature death or severe motor disability. New therapies have been developed in recent years that change the natural history of the disease. The aim of this study is to describe patients included in the Polish Registry of SMA, with a focus on the course of type 3 SMA (SMA3) before the availability of disease-modifying treatments. RESULTS: 790 patients with SMA were included in the registry (173 with type 1 [SMA1], 218 with type 2 [SMA2], 393 with SMA3, and six with type 4 SMA [SMA4]), most (52%) of whom were adults. Data on SMN2 gene copy number were available for 672 (85%) patients. The mean age of onset was 5 months for SMA1, 11.5 months for SMA2, and 4.5 years for SMA3. In patients with SMA3, the first symptoms occurred earlier in those with three copies of SMN2 than in those with four copies of SMN2 (3.2 years vs. 6.7 years). The age of onset of SMA3 was younger in girls than in boys (3.1 years vs. 5.7 years), with no new cases observed in women older than 16 years. Male patients outnumbered female patients, especially among patients with SMA3b (49 female vs. 85 male patients) and among patients with SMA3 with four copies of SMN2 (30 female vs. 69 male patients). 44% of patients with SMA3 were still able to walk; in those who were not still able to walk, the mean age of immobilization was 14.0 years. Patients with SMA3a (age of onset < 3 years) and three copies of SMN2 had significantly worse prognosis for remaining ambulant than patients with SMA3b (age of onset ≥ 3 years) and four copies of SMN2. CONCLUSIONS: The Registry of SMA is an effective tool for assessing the disease course in the real world setting. SMN2 copy number is an important prognostic factor for the age of onset and ambulation in SMA3. Sex and age of disease onset also strongly affect the course of SMA. Data supplied by this study can aid treatment decisions.

Clinical and molecular characterization of craniofrontonasal syndrome: new symptoms and novel pathogenic variants in the EFNB1 gene.

BACKGROUND: Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder that results from pathogenic variants in the EFNB1 gene. The syndrome paradoxically presents with greater severity of the symptoms in heterozygous females than hemizygous males. RESULTS: We have recruited and screened a female cohort affected with CFNS. Our primary finding was the description of monozygotic twins, i.e., patients 5 and 6, discordant for the CFNS phenotype. Intriguingly, patient 5 presented classical CFNS gestalt, whereas patient 6 manifested only very subtle craniofacial features, not resembling CFNS. Besides, we have expanded the mutational spectrum of the EFNB1 gene through reporting four novel pathogenic variants-p.(Trp12*), p.(Cys64Phe), p.(Tyr73Metfs*86), p.(Glu210*). All those alterations were found applying either targeted NGS of a custom gene panel or PCR followed by Sanger sequencing and evaluated using in silico predictors. Lastly, we have also expanded the CFNS phenotypic spectrum by describing in patient 3 several novel features of the syndrome, such as bifid hallux, bicornuate uterus, and abnormal right ovary segmented into six parts. CONCLUSIONS: We have described the unreported so far differences of the clinical phenotype in the monozygotic twin patients 5 and 6 harboring an identical p.(Glu210*) variant located in the EFNB1 gene. With our finding, we have pointed to an unusual phenomenon of mildly affected females with CFNS, who may not manifest features suggestive of the syndrome. Consequently, this study may be valuable for geneticists consulting patients with craniofacial disorders.

Pathogenic Mutations and Putative Phenotype-Affecting Variants in Polish Myofibrillar Myopathy Patients.

Myofibrillar myopathies (MFM) are heterogeneous hereditary muscle diseases with characteristic myopathological features of Z-disk dissolution and aggregates of its degradation products. The onset and progression of the disease are variable, with an elusive genetic background, and around half of the cases lacking molecular diagnosis. Here, we attempted to establish possible genetic foundations of MFM by performing whole exome sequencing (WES) in eleven unrelated families of 13 patients clinically diagnosed as MFM spectrum. A filtering strategy aimed at identification of variants related to the disease was used and included integrative analysis of WES data and human phenotype ontology (HPO) terms, analysis of muscle-expressed genes, and analysis of the disease-associated interactome. Genetic diagnosis was possible in eight out of eleven cases. Putative causative mutations were found in the DES (two cases), CRYAB, TPM3, and SELENON (four cases) genes, the latter typically presenting with a rigid spine syndrome. Moreover, a variety of additional, possibly phenotype-affecting variants were found. These findings indicate a markedly heterogeneous genetic background of MFM and show the usefulness of next generation sequencing in the identification of disease-associated mutations. Finally, we discuss the emerging concept of variant load as the basis of phenotypic heterogeneity.

Floppy infant syndrome as a first manifestation of LMNA-related congenital muscular dystrophy.

LMNA-related congenital muscular dystrophy (L-CMD) is the most severe phenotypic form of skeletal muscle laminopathies. This paper reports clinical presentation of the disease in 15 Polish patients from 13 families with genetically confirmed skeletal muscle laminopathy. In all these patients floppy infant syndrome was the first manifestation of the disease. The genetic diagnosis was established by next generation sequencing (targeted panel or exome; 11 patients) or classic Sanger sequencing (4 patients). In addition to known pathogenic LMNA variants: c.116A > G (p.Asn39Ser), c.745C > T (p.Arg249Trp), c.746G > A (p.Arg249Gln), c.1072G > A (p.Glu358Lys), c.1147G > A (p.Glu383Lys), c.1163G > C (p.Arg388Pro), c.1357C > T (p.Arg453Trp), c.1583C > G (p.Thr528Arg), we have identified three novel ones: c.121C > G (p.Arg41Gly), c.1127A > G (p.Tyr376Cys) and c.1160T > C (p.Leu387Pro). Eleven patients had de novo mutations, 4 - familial. In one family we observed intrafamilial variability of clinical course: severe L-CMD in the male proband, intermediate form in his sister and asymptomatic in their mother. One asymptomatic father had somatic mosaicism. L-CMD should be suspected in children with hypotonia in infancy and delayed motor development, who have poor head control, severe hyperlordosis and unstable and awkward gait. Serum creatine kinase may be high (~1000IU/l). Progression of muscle weakness is fast, leading to early immobilization. In some patients with L-CMD joint contractures can develop with time. MRI shows that the most frequently affected muscles are the serratus anterior, lumbar paraspinal, gluteus, vastus, adductor magnus, hamstrings, medial head of gastrocnemius and soleus. Ultra-rare laminopathies can be a relatively common cause of generalized hypotonia in children. Introduction of wide genome sequencing methods was a breakthrough in diagnostics of diseases with great clinical and genetic variability and allowed approach "from genotype do phenotype". However target sequencing of LMNA gene could be considered in selected patients with clinical picture suggestive for laminopathy.

Case Report: Further Delineation of Neurological Symptoms in Young Children Caused by Compound Heterozygous Mutation in the PIEZO2 Gene.

PIEZO2 protein is a unique ion channel that converts mechanical impulses into cellular signals in somatosensory neurons and is involved in various mechanotransduction pathways. The recessive PIEZO2 loss-of-function pathogenic variants are associated with distal arthrogryposis with impaired proprioception and touch (DAIPT). Here we present three new DAIPT patients. The genetic diagnosis was established by exome sequencing and let us to identify 6 novel loss-of-function PIEZO2 variants: four splicing (c.1080+1G>A, c.4092+1G>T, c.6355+1G>T, and c.7613+1G>A), one nonsense (c.6088C>T) and one frameshift variant (c.6175_6191del) for which mosaic variant was identified in proband's mother. All patients presented typical symptoms at birth, with congenital contractures, bilateral hip dislocation/dysplasia, generalized hypotonia, transient feeding and difficulties. Two were afflicted by transient respiratory insufficiency. In all children motor development was severely delayed. In one patient, severe cognitive delay was also observed. Moreover, among the cases described by us there is the youngest diagnosed child to date.

Incidence of spinal muscular atrophy in Poland--more frequent than predicted?

BACKGROUND: The application of molecular methods has enhanced and enlarged the diagnostics of spinal muscular atrophy (SMA) and its carriership. It allows for reliable epidemiological studies which are of importance to demography and genetic counseling. METHODS: This study sought to evaluate the incidence of SMA in Poland, on the basis of the prevalence of the SMN1 gene deletion carrier state in the general population, as well as an analysis of all cases of SMA diagnosed in the years 1998-2005. RESULTS: The prevalence of the SMN1 gene deletion carrier state was estimated at 1 per 35 persons (17/600), yielding an incidence of SMA equal to 1 per 4,900. By contrast, the incidence of SMA based on the results of the meta-analysis was an estimated 1 per 7,127 in Warsaw and 1 per 9,320 persons across Poland, suggesting that some cases of SMA remain undiagnosed. SMA1 predominated among the diagnoses, accounting for 69% of all cases. CONCLUSION: A high prevalence of the SMN1 deletion carrier state in the general population indicates that SMA could be a more frequent disease than is predicted by the epidemiological data regarding diagnosed cases.

Advances in Newborn Screening and Presymptomatic Diagnosis of Spinal Muscular Atrophy.

Spinal muscular atrophy 5q (SMA5q) is one of the most severe and common genetic diseases. In the natural course, the disease leads to premature death (in acute forms) or severe motor disability (in chronic forms). As the genetic basis of SMA is very homogenous, the diagnostics are based entirely on simple and sensitive genetic testing. In the last few years, innovative methods of therapy have been developed based on SMN2 gene modification, such as splicing, or replacement of the damaged SMN1 gene (gene therapy). Although these approaches have shown high efficacy, results depend on the age/disease stage at which therapy is initiated. The best results have been obtained in presymptomatic patients. Indeed, introduction of therapy in the pre- or early symptomatic stage of the disease seems to be crucial for maximizing effects. Thus, all the criteria for the implementation of neonatal screening for SMA have been met, and many countries, ie, the USA, Germany, Belgium, and Australia, have started NBS national/pilot programs for SMA. The initial results of these programs indicate a high frequency of the disease, reaching 1 per 7 thousand live births in Europe, as well as early symptomatology (first weeks of life in severe cases) and a high frequency of patients with 4 SMN2 copies. Overall, the time for therapy inclusion in patients with 4 SMN2 copies remain under discussion. More precise predictors/biomarkers of the clinical course are needed. At the same time, it seems advisable to offer other solutions, such as population carrier screening. As the long-term effects of different treatments on the natural history of SMA are unknown, the natural history of the disease needs to be re-evaluated.

The phenotype-driven computational analysis yields clinical diagnosis for patients with atypical manifestations of known intellectual disability syndromes.

BACKGROUND: Due to extensive clinical and genetic heterogeneity of intellectual disability (ID) syndromes, the process of diagnosis is very challenging even for expert clinicians. Despite recent advancements in molecular diagnostics methodologies, a significant fraction of ID patients remains without a clinical diagnosis. METHODS, RESULTS, AND CONCLUSIONS: Here, in a prospective study on a cohort of 21 families (trios) with a child presenting with ID of unknown etiology, we executed phenotype-driven bioinformatic analysis method, PhenIX, utilizing targeted next-generation sequencing (NGS) data and Human Phenotype Ontology (HPO)-encoded phenotype data. This approach resulted in clinical diagnosis for eight individuals presenting with atypical manifestations of Rubinstein-Taybi syndrome 2 (MIM 613684), Spastic Paraplegia 50 (MIM 612936), Wiedemann-Steiner syndrome (MIM 605130), Cornelia de Lange syndrome 2 (MIM 300590), Cerebral creatine deficiency syndrome 1 (MIM 300352), Glass Syndrome (MIM 612313), Mental retardation, autosomal dominant 31 (MIM 616158), and Bosch-Boonstra-Schaaf optic atrophy syndrome (MIM 615722).

Phenotype modifiers of spinal muscular atrophy: the number of SMN2 gene copies, deletion in the NAIP gene and probably gender influence the course of the disease.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations of the SMN1 gene. It is characterized by significant phenotype variability. In this study, we analyzed possible phenotype modifiers of the disease - the size of the deletion in the SMA region, the number of SMN2 gene copies, as well as the effect of gender. Among the factors analyzed, two seem to influence the SMA phenotype: the number of SMN2 gene copies and a deletion in the NAIP gene. A higher number of SMN2 copies makes the clinical symptoms more benign, and the NAIP gene deletion is associated with a more severe phenotype. The influence of gender remains unclear. In a group of 1039 patients, 55% of whom were male, the greatest disproportion was in the SMA1 (F/M = 0.78) and SMA3b (F/M = 0.45) forms. In SMA1 a deletion in the NAIP gene was seen twice as frequently in girls compared to boys. In three patients, we observed genotypes atypical for the chronic forms of SMA: two patients with SMA3a and 3b had a deletion of the NAIP gene, and a third patient with SMA2 had one copy of the SMN2 gene.

Novel variant in HDAC8 gene resulting in the severe Cornelia de Lange phenotype.

Cornelia de Lange syndrome (CDLS) is a clinically and genetically heterogeneous developmental disorder characterized by multiple malformations. Primarily, affected individuals have unique and recognizable dysmorphic facial features, cleft palate, distal limb defects, growth retardation, and developmental delay. However, also milder, as well as slightly phenotypically different forms exist. We described herein a patient with CDLS5, an X-linked form, caused by mutations in the HDAC8 gene inherited form the mosaic mother. Analysis of results from whole exome sequencing identified two variants with possible impact on the phenotype. Of them, hemizygous variant (c.938G>A, p.Arg313Gln) inherited from the mosaic mother, was further proved to lead to disease in the proband. Our intention was to delineate this syndrome but also point out the clinical course of the disease, which only in combination with a facial phenotype allow for verification of exome sequencing result.

Unaffected patients with a homozygous absence of the SMN1 gene.

In this report, we present three families in which we identified asymptomatic carriers of a homozygous absence of the SMN1 gene. In the first family, the bialleleic deletion was found in three of four siblings: two affected brothers (SMA type 3a and 3b) and a 25-years-old asymptomatic sister. All of them have four SMN2 copies. In the second family, four of six siblings are affected (three suffer from SMA2 and one from SMA3a), each with three SMN2 copies. The clinically asymptomatic 47-year-old father has the biallelic deletion and four SMN2 copies. In the third family, the biallelic SMN1 absence was found in a girl affected with SMA1 and in her healthy 53-years-old father who had five SMN2 copies. Our findings as well as those of other authors show that an increased number of SMN2 copies in healthy carriers of the biallelic SMN1 deletion is an important SMA phenotype modifier, but probably not the only one.

Nonsense-mediated messenger RNA decay of survival motor neuron 1 causes spinal muscular atrophy.

Autosomal recessive proximal spinal muscular atrophy (SMA) is a neurodegenerative disorder resulting from functional loss of survival motor neuron 1 (SMN1). Homozygous absence of SMN1 due to deletion or gene conversion accounts for about 96% of SMA cases. In the remaining 4%, subtle SMN1 mutations are commonly identified. Here, we describe two novel intragenic SMN1 mutations in three type I SMA individuals: a point mutation in exon 3 (c.469C > T) and a substitution in intron 4 (c.628-140A > G). In-vivo splicing assays demonstrated that the intronic substitution creates a novel splice donor site, culminating in aberrant splicing and insertion of 65 bp from intron 4 between exons 4 and 5 in SMN1 transcripts (c.627_628ins65). Both mutations render SMN1 transcripts susceptible to nonsense-mediated mRNA decay (NMD), resulting in mRNA degradation, insufficient SMN protein levels and development of an SMA phenotype. Treatment of patient cell lines with the translation inhibitors puromycin and emetine markedly increased the levels of mutant SMN1 transcripts. A similar effect was observed after siRNA-mediated knockdown of UPF1, a factor essential for NMD. This study provides first evidence that NMD of SMN1 transcripts is responsible for the molecular basis of disease in a subset of SMA patients.