Physical performance and plasma metabolic profile as potential prognostic factors in metastatic lung cancer patients.

BACKGROUND: Low physical performance is associated with higher mortality rate in multiple pathological conditions. Here, we aimed to determine whether body composition and physical performance could be prognostic factors in non-small cell lung cancer (NSCLC) patients. Moreover, we performed an exploratory approach to determine whether plasma samples from NSCLC patients could directly affect metabolic and structural phenotypes in primary muscle cells. METHODS: This prospective cohort study included 55 metastatic NSCLC patients and seven age-matched control subjects. Assessments included physical performance, body composition, quality of life and overall survival rate. Plasma samples from a sub cohort of 18 patients were collected for exploratory studies in cell culture and metabolomic analysis. RESULTS: We observed a higher survival rate in NSCLC patients with high performance in the timed up-and-go (+320%; p = .007), sit-to-stand (+256%; p = .01) and six-minute walking (+323%; p = .002) tests when compared to NSCLC patients with low physical performance. There was no significant association for similar analysis with body composition measurements (p > .05). Primary human myotubes incubated with plasma from NSCLC patients with low physical performance had impaired oxygen consumption rate (-54.2%; p < .0001) and cell proliferation (-44.9%; p = .007). An unbiased metabolomic analysis revealed a list of specific metabolites differentially expressed in the plasma of NSCLC patients with low physical performance. CONCLUSION: These novel findings indicate that physical performance is a prognostic factor for overall survival in NSCLC patients and provide novel insights into circulating factors that could impair skeletal muscle metabolism.

Continued benefit of nusinersen initiated in the presymptomatic stage of spinal muscular atrophy: 5-year update of the NURTURE study.

INTRODUCTION/AIMS: NURTURE (NCT02386553) is an open-label study of nusinersen in children (two SMN2 copies, n = 15; three SMN2 copies, n = 10) who initiated treatment in the presymptomatic stage of spinal muscular atrophy (SMA). A prior analysis after ~3 y showed benefits on survival, respiratory outcomes, motor milestone achievement, and a favorable safety profile. An additional 2 y of follow-up (data cut: February 15, 2021) are reported. METHODS: The primary endpoint is time to death or respiratory intervention (≥6 h/day continuously for ≥7 days or tracheostomy). Secondary outcomes include overall survival, motor function, and safety. RESULTS: Median age of children was 4.9 (3.8-5.5) y at last visit. No children have discontinued the study or treatment. All were alive. No additional children utilized respiratory intervention (defined per primary endpoint) since the prior data cut. Children with three SMN2 copies achieved all World Health Organization (WHO) motor milestones, with all but one milestone in one child within normal developmental timeframes. All 15 children with two SMN2 copies achieved sitting without support, 14/15 walking with assistance, and 13/15 walking alone. Mean Hammersmith Functional Motor Scale Expanded total scores showed continued improvement. Subgroups with two SMN2 copies, minimum baseline compound muscle action potential amplitude ≥2 mV, and no baseline areflexia had better motor and nonmotor outcomes versus all children with two SMN2 copies. DISCUSSION: These results demonstrate the value of early treatment, durability of treatment effect, and favorable safety profile after ~5 y of nusinersen treatment. Inclusion/exclusion criteria and baseline characteristics should be considered when interpreting presymptomatic SMA trial data.

Cancer-induced muscle atrophy is determined by intrinsic muscle oxidative capacity.

We tested the hypothesis that cancer cachexia progression would induce oxidative post-translational modifications (Ox-PTMs) associated with skeletal muscle wasting, with different responses in muscles with the prevalence of glycolytic and oxidative fibers. We used cysteine-specific isotopic coded affinity tags (OxICAT) and gel-free mass spectrometry analysis to investigate the cysteine Ox-PTMs profile in the proteome of both plantaris (glycolytic) and soleus (oxidative) muscles in tumor-bearing and control rats. Histological analysis revealed muscle atrophy in type II fibers in plantaris muscle, with no changes in plantaris type I fibers and no differences in both soleus type I and II fibers in tumor-bearing rats when compared to healthy controls. Tumor progression altered the Ox-PTMs profile in both plantaris and soleus. However, pathway analysis including the differentially oxidized proteins revealed tricarboxylic acid cycle and oxidative phosphorylation as main affected pathways in plantaris muscle from tumor-bearing rats, while the same analysis did not show main metabolic pathways affected in the soleus muscle. In addition, cancer progression affected several metabolic parameters such as ATP levels and markers of oxidative stress associated with muscle atrophy in plantaris muscle, but not in soleus. However, isolated soleus from tumor-bearing rats had a reduced force production capacity when compared to controls. These novel findings demonstrate that tumor-bearing rats have severe muscle atrophy exclusively in glycolytic fibers. Cancer progression is associated with cysteine Ox-PTMs in the skeletal muscle, but these modifications affect different pathways in a glycolytic muscle compared to an oxidative muscle, indicating that intrinsic muscle oxidative capacity determines the response to cancer cachectic effects.

Muscle microRNAs in the cerebrospinal fluid predict clinical response to nusinersen therapy in type II and type III spinal muscular atrophy patients.

BACKGROUND AND PURPOSE: The antisense oligonucleotide nusinersen (Spinraza) regulates splicing of the survival motor neuron 2 (SMN2) messenger RNA to increase SMN protein expression. Nusinersen has improved ventilator-free survival and motor function outcomes in infantile onset forms of spinal muscular atrophy (SMA), treated early in the course of the disease. However, the response in later onset forms of SMA is highly variable and dependent on symptom severity and disease duration at treatment initiation. Therefore, we aimed to identify novel noninvasive biomarkers that could predict the response to nusinersen in type II and III SMA patients. METHODS: Thirty-four SMA patients were included. We applied next generation sequencing to identify microRNAs in the cerebrospinal fluid (CSF) as candidate biomarkers predicting response to nusinersen. Hammersmith Functional Motor Scale Expanded (HFMSE) was conducted at baseline and 6 months after initiation of nusinersen therapy to assess motor function. Patients changing by ≥3 or ≤0 points in the HFMSE total score were considered to be responders or nonresponders, respectively. RESULTS: Lower baseline levels of two muscle microRNAs (miR-206 and miR-133a-3p), alone or in combination, predicted the clinical response to nusinersen after 6 months of therapy. Moreover, miR-206 levels were inversely correlated with the HFMSE score. CONCLUSIONS: Lower miR-206 and miR-133a-3p in the CSF predict more robust clinical response to nusinersen treatment in later onset SMA patients. These novel findings have high clinical relevance for identifying early treatment response to nusinersen in later onset SMA patients and call for testing the ability of miRNAs to predict more sustained long-term benefit.

Prion protein quantification in human cerebrospinal fluid as a tool for prion disease drug development.

Reduction of native prion protein (PrP) levels in the brain is an attractive strategy for the treatment or prevention of human prion disease. Clinical development of any PrP-reducing therapeutic will require an appropriate pharmacodynamic biomarker: a practical and robust method for quantifying PrP, and reliably demonstrating its reduction in the central nervous system (CNS) of a living patient. Here we evaluate the potential of ELISA-based quantification of human PrP in human cerebrospinal fluid (CSF) to serve as a biomarker for PrP-reducing therapeutics. We show that CSF PrP is highly sensitive to plastic adsorption during handling and storage, but its loss can be minimized by the addition of detergent. We find that blood contamination does not affect CSF PrP levels, and that CSF PrP and hemoglobin are uncorrelated, together suggesting that CSF PrP is CNS derived, supporting its relevance for monitoring the tissue of interest and in keeping with high PrP abundance in brain relative to blood. In a cohort with controlled sample handling, CSF PrP exhibits good within-subject test-retest reliability (mean coefficient of variation, 13% in samples collected 8-11 wk apart), a sufficiently stable baseline to allow therapeutically meaningful reductions in brain PrP to be readily detected in CSF. Together, these findings supply a method for monitoring the effect of a PrP-reducing drug in the CNS, and will facilitate development of prion disease therapeutics with this mechanism of action.

Detection of SMN1 to SMN2 gene conversion events and partial SMN1 gene deletions using array digital PCR.

Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutations of survival motor neuron 1 (SMN1) but retention of one or more copies of the paralog SMN2. Within the SMA population, there is substantial variation in SMN2 copy number (CN); in general, those individuals with SMA who have a high SMN2 CN have a milder disease. Because SMN2 functions as a disease modifier, its accurate CN determination may have clinical relevance. In this study, we describe the development of array digital PCR (dPCR) to quantify SMN1 and SMN2 CNs in DNA samples using probes that can distinguish the single nucleotide difference between SMN1 and SMN2 in exon 8. This set of dPCR assays can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 CN and disease severity. We can detect SMN1-SMN2 gene conversion events in DNA samples by comparing CNs at exon 7 and exon 8. Partial deletions of SMN1 can also be detected with dPCR by comparing CNs at exon 7 or exon 8 with those at intron 1. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 CNs from SMA samples as well as identify gene conversion events and partial deletions of SMN1.

Neuronal modeling of alternating hemiplegia of childhood reveals transcriptional compensation and replicates a trigger-induced phenotype.

Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disease caused by heterozygous de novo missense mutations in the ATP1A3 gene that encodes the neuronal specific α3 subunit of the Na,K-ATPase (NKA) pump. Mechanisms underlying patient episodes including environmental triggers remain poorly understood, and there are no empirically proven treatments for AHC. In this study, we generated patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls for the E815K ATP1A3 mutation that causes the most phenotypically severe form of AHC. Using an in vitro iPSC-derived cortical neuron disease model, we found elevated levels of ATP1A3 mRNA in AHC lines compared to controls, without significant perturbations in protein expression. Microelectrode array analyses demonstrated that in cortical neuronal cultures, ATP1A3+/E815K iPSC-derived neurons displayed less overall activity than neurons differentiated from isogenic mutation-corrected and unrelated control cell lines. However, induction of cellular stress by elevated temperature revealed a hyperactivity phenotype following heat stress in ATP1A3+/E815K neurons compared to control lines. Treatment with flunarizine, a drug commonly used to prevent AHC episodes, did not impact this stress-triggered phenotype. These findings support the use of iPSC-derived neuronal cultures for studying complex neurodevelopmental conditions such as AHC and provide a platform for mechanistic discovery in a human disease model.

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis.

Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca2+-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.

Whole blood survival motor neuron protein levels correlate with severity of denervation in spinal muscular atrophy.

INTRODUCTION: We sought to determine whether survival motor neuron (SMN) protein blood levels correlate with denervation and SMN2 copies in spinal muscular atrophy (SMA). METHODS: Using a mixed-effect model, we tested associations between SMN levels, compound muscle action potential (CMAP), and SMN2 copies in a cohort of 74 patients with SMA. We analyzed a subset of 19 of these patients plus four additional patients who had been treated with received gene therapy to examine SMN trajectories early in life. RESULTS: Patients with SMA who had lower CMAP values had lower circulating SMN levels (P = .04). Survival motor neuron protein levels were different between patients with two and three SMN2 copies (P < .0001) and between symptomatic and presymptomatic patients (P < .0001), with the highest levels after birth and progressive decline over the first 3 years. Neither nusinersen nor gene therapy clearly altered SMN levels. DISCUSSION: These data provide evidence that whole blood SMN levels correlate with SMN2 copy number and severity of denervation.

Stander Use in Spinal Muscular Atrophy: Results From a Large Natural History Database.

PURPOSE: The purpose of this study was to describe stander use in a natural history cohort of drug therapy-naïve children with spinal muscular atrophy (SMA) who are not walking and identify factors associated with consistent stander use. METHODS: Data from 397 children with SMA types 1 and 2 characterized the prevalence and frequency of stander use. Predictors of consistent stander use explored were SMA type, survival motor neuron 2 gene (SMN2) copy number, respiratory support, and motor performance. RESULTS: Prevalence of consistent stander use was 13% in type 1 and 68% in type 2. SMA type, SMN2 copy number, respiratory support, and head rotation control each predicted consistent stander use. CONCLUSIONS: Findings characterize stander use in children with SMA who are not walking, address important safety considerations, identify factors that may inform physical therapists' clinical decision-making related to standing program prescription, and provide guidance for future prospective studies.

Complete sequencing of the SMN2 gene in SMA patients detects SMN gene deletion junctions and variants in SMN2 that modify the SMA phenotype.

Spinal muscular atrophy (SMA) is a progressive motor neuron disease caused by loss or mutation of the survival motor neuron 1 (SMN1) gene and retention of SMN2. We performed targeted capture and sequencing of the SMN2, CFTR, and PLS3 genes in 217 SMA patients. We identified a 6.3 kilobase deletion that occurred in both SMN1 and SMN2 (SMN1/2) and removed exons 7 and 8. The deletion junction was flanked by a 21 bp repeat that occurred 15 times in the SMN1/2 gene. We screened for its presence in 466 individuals with the known SMN1 and SMN2 copy numbers. In individuals with 1 SMN1 and 0 SMN2 copies, the deletion occurred in 63% of cases. We modeled the deletion junction frequency and determined that the deletion occurred in both SMN1 and SMN2. We have identified the first deletion junction where the deletion removes exons 7 and 8 of SMN1/2. As it occurred in SMN1, it is a pathogenic mutation. We called variants in the PLS3 and SMN2 genes, and tested for association with mild or severe exception patients. The variants A-44G, A-549G, and C-1897T in intron 6 of SMN2 were significantly associated with mild exception patients, but no PLS3 variants correlated with severity. The variants occurred in 14 out of 58 of our mild exception patients, indicating that mild exception patients with an intact SMN2 gene and without modifying variants occur. This sample set can be used in the association analysis of candidate genes outside of SMN2 that modify the SMA phenotype.

Direct evidence of impaired neuronal Na/K-ATPase pump function in alternating hemiplegia of childhood.

Mutations in ATP1A3 encoding the catalytic subunit of the Na/K-ATPase expressed in mammalian neurons cause alternating hemiplegia of childhood (AHC) as well as an expanding spectrum of other neurodevelopmental syndromes and neurological phenotypes. Most AHC cases are explained by de novo heterozygous ATP1A3 mutations, but the fundamental molecular and cellular consequences of these mutations in human neurons are not known. In this study, we investigated the electrophysiological properties of neurons generated from AHC patient-specific induced pluripotent stem cells (iPSCs) to ascertain functional disturbances underlying this neurological disease. Fibroblasts derived from two subjects with AHC, a male and a female, both heterozygous for the common ATP1A3 mutation G947R, were reprogrammed to iPSCs. Neuronal differentiation of iPSCs was initiated by neurogenin-2 (NGN2) induction followed by co-culture with mouse glial cells to promote maturation of cortical excitatory neurons. Whole-cell current clamp recording demonstrated that, compared with control iPSC-derived neurons, neurons differentiated from AHC iPSCs exhibited a significantly lower level of ouabain-sensitive outward current ('pump current'). This finding correlated with significantly depolarized potassium equilibrium potential and depolarized resting membrane potential in AHC neurons compared with control neurons. In this cellular model, we also observed a lower evoked action potential firing frequency when neurons were held at their resting potential. However, evoked action potential firing frequencies were not different between AHC and control neurons when the membrane potential was clamped to -80 mV. Impaired neuronal excitability could be explained by lower voltage-gated sodium channel availability at the depolarized membrane potential observed in AHC neurons. Our findings provide direct evidence of impaired neuronal Na/K-ATPase ion transport activity in human AHC neurons and demonstrate the potential impact of this genetic defect on cellular excitability.

Emerging therapies and challenges in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a hereditary neurodegenerative disease with severity ranging from progressive infantile paralysis and premature death (type I) to limited motor neuron loss and normal life expectancy (type IV). Without disease-modifying therapies, the impact is profound for patients and their families. Improved understanding of the molecular basis of SMA, disease pathogenesis, natural history, and recognition of the impact of standardized care on outcomes has yielded progress toward the development of novel therapeutic strategies and are summarized. Therapeutic strategies in the pipeline are appraised, ranging from SMN1 gene replacement to modulation of SMN2 encoded transcripts, to neuroprotection, to an expanding repertoire of peripheral targets, including muscle. With the advent of preliminary trial data, it can be reasonably anticipated that the SMA treatment landscape will transform significantly. Advancement in presymptomatic diagnosis and screening programs will be critical, with pilot newborn screening studies underway to facilitate preclinical diagnosis. The development of disease-modifying therapies will necessitate monitoring programs to determine the long-term impact, careful evaluation of combined treatments, and further acceleration of improvements in supportive care. In advance of upcoming clinical trial results, we consider the challenges and controversies related to the implementation of novel therapies for all patients and set the scene as the field prepares to enter an era of novel therapies. Ann Neurol 2017;81:355-368.

Natural history of infantile-onset spinal muscular atrophy.

OBJECTIVE: Infantile-onset spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality, typically resulting in death preceding age 2. Clinical trials in this population require an understanding of disease progression and identification of meaningful biomarkers to hasten therapeutic development and predict outcomes. METHODS: A longitudinal, multicenter, prospective natural history study enrolled 26 SMA infants and 27 control infants aged <6 months. Recruitment occurred at 14 centers over 21 months within the NINDS-sponsored NeuroNEXT (National Network for Excellence in Neuroscience Clinical Trials) Network. Infant motor function scales (Test of Infant Motor Performance Screening Items [TIMPSI], The Children's Hospital of Philadelphia Infant Test for Neuromuscular Disorders, and Alberta Infant Motor Score) and putative physiological and molecular biomarkers were assessed preceding age 6 months and at 6, 9, 12, 18, and 24 months with progression, correlations between motor function and biomarkers, and hazard ratios analyzed. RESULTS: Motor function scores (MFS) and compound muscle action potential (CMAP) decreased rapidly in SMA infants, whereas MFS in all healthy infants rapidly increased. Correlations were identified between TIMPSI and CMAP in SMA infants. TIMPSI at first study visit was associated with risk of combined endpoint of death or permanent invasive ventilation in SMA infants. Post-hoc analysis of survival to combined endpoint in SMA infants with 2 copies of SMN2 indicated a median age of 8 months at death (95% confidence interval, 6, 17). INTERPRETATION: These data of SMA and control outcome measures delineates meaningful change in clinical trials in infantile-onset SMA. The power and utility of NeuroNEXT to provide "real-world," prospective natural history data sets to accelerate public and private drug development programs for rare disease is demonstrated. Ann Neurol 2017;82:883-891.

Clinical trial of L-Carnitine and valproic acid in spinal muscular atrophy type I.

INTRODUCTION: The aim of this study was to determine the safety and therapeutic potential of L-carnitine and valproic acid (VPA) in infants with spinal muscular atrophy (SMA). METHODS: Our investigation was an open-label phase 2 multicenter trial of L-carnitine and VPA in infants with SMA type I with retrospective comparison to an untreated, matched cohort. Primary outcomes were: safety and adverse events; secondary outcomes were survival, time to death/>16 hours/day of ventilator support; motor outcomes; and maximum ulnar compound motor action potential amplitude. RESULTS: A total of 245 AEs were observed in 35 of the 37 treated subjects (95%). Respiratory events accounted for 49% of all adverse events, resulting in 14 deaths. Survival was not significantly different between treated and untreated cohorts. DISCUSSION: This trial provides evidence that, in infants with SMA type I, L-carnitine/VPA is ineffective at altering survival. The substantial proportion of infants reaching end-points within 6 months of enrollment underscores the urgent need for pre-symptomatic treatment in SMA type I. Muscle Nerve 57: 193-199, 2018.

Astrocytes influence the severity of spinal muscular atrophy.

Systemically low levels of survival motor neuron-1 (SMN1) protein cause spinal muscular atrophy (SMA). α-Motor neurons of the spinal cord are considered particularly vulnerable in this genetic disorder and their dysfunction and loss cause progressive muscle weakness, paralysis and eventually premature death of afflicted individuals. Historically, SMA was therefore considered a motor neuron-autonomous disease. However, depletion of SMN in motor neurons of normal mice elicited only a very mild phenotype. Conversely, restoration of SMN to motor neurons in an SMA mouse model had only modest effects on the SMA phenotype and survival. Collectively, these results suggested that additional cell types contribute to the pathogenesis of SMA, and understanding the non-autonomous requirements is crucial for developing effective therapies. Astrocytes are critical for regulating synapse formation and function as well as metabolic support for neurons. We hypothesized that astrocyte functions are disrupted in SMA, exacerbating disease progression. Using viral-based restoration of SMN specifically to astrocytes, survival in severe and intermediate SMA mice was observed. In addition, neuromuscular circuitry was improved. Astrogliosis was prominent in end-stage SMA mice and in post-mortem patient spinal cords. Increased expression of proinflammatory cytokines was partially normalized in treated mice, suggesting that astrocytes contribute to the pathogenesis of SMA.

Survival of motor neurone protein is required for normal postnatal development of the spleen.

Spinal muscular atrophy (SMA), traditionally described as a predominantly childhood form of motor neurone disease, is the leading genetic cause of infant mortality. Although motor neurones are undoubtedly the primary affected cell type, the severe infantile form of SMA (Type I SMA) is now widely recognised to represent a multisystem disorder where a variety of organs and systems in the body are also affected. Here, we report that the spleen is disproportionately small in the 'Taiwanese' murine model of severe SMA (Smn-/- ;SMN2tg/0 ), correlated to low levels of cell proliferation and increased cell death. Spleen lacks its distinctive red appearance and presents with a degenerated capsule and a disorganised fibrotic architecture. Histologically distinct white pulp failed to form and this was reflected in an almost complete absence of B lymphocytes necessary for normal immune function. In addition, megakaryoctyes persisted in the red pulp. However, the vascular density remained unchanged in SMA spleen. Assessment of the spleen in SMA patients with the infantile form of the disease indicated a range of pathologies. We conclude that development of the spleen fails to occur normally in SMA mouse models and human patients. Thus, further analysis of immune function is likely to be required to fully understand the full extent of systemic disease pathology in SMA.

Pregnancy and delivery in women with spinal muscular atrophy.

OBJECTIVES: To expand the limited available knowledge about pregnancy and delivery in women with spinal muscular atrophy (SMA) using a cohort of genetically proven SMA patients from USA. METHODS: This was a cross-sectional questionnaire-based study. We mailed questionnaires to 58 women with confirmed SMA. RESULTS: Thirty-two women responded, reporting 35 pregnancies, including 19 women with at least one pregnancy. In this cohort, preterm labor and delivery by cesarean section were more common in mothers with SMA particularly SMA type 2. Seventy-four percent of mothers reported increased weakness during pregnancy that persisted after delivery in 42%. SMA mothers generally had a positive experience and good outcomes and elected to have more than one pregnancy. CONCLUSION: This information regarding pregnancy in women with genetically confirmed 5q SMA will prove useful in guiding future research and in providing counseling to women with SMA.

Clinical phenotypes as predictors of the outcome of skipping around DMD exon 45.

OBJECTIVE: Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion-exon 45 (Δ45)-may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients. METHODS: Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy were analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions. RESULTS: As expected, deletions of either exons 45 to 47 (Δ45-47) or exons 45 to 48 (Δ45-48) result in BMD in 97% (36 of 37) of subjects. Unexpectedly, deletion of exons 45 to 46 (Δ45-46) is associated with the more severe DMD phenotype in 4 of 4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44 to 45 (Δ44-45) were found within the United Dystrophinopathy Project database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype. INTERPRETATION: The observation that Δ45-46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of exon 44 and multiexon skipping of exons 46 and 47 (or exons 46-48) are better potential therapies than skipping of exon 46 alone.