A homozygous canonical splice acceptor site mutation in PRUNE1 is responsible for a rare childhood neurodegenerative disease in Manitoba Cree families.

Autosomal recessive PRUNE1 mutations are reported to cause a severe neurodevelopmental disorder with microcephaly, hypotonia, and brain malformations. We describe clinical and neuropathological features in a cohort of nine individuals of Cree descent who, because of a founder effect, are homozygous for the same PRUNE1 mutation. They follow the course of a combined neuromuscular and neurodegenerative disease, rather than a pure failure of normal development. This cohort presented in infancy with features of lower motor neuron disease, such as hypotonia, contractures, tongue fasciculations, and feeding difficulties in the absence of congenital brain anomalies and microcephaly. A neurodegenerative course followed with onset of seizures, spasticity, and respiratory insufficiency. Muscle biopsies showed denervation/reinnervation features, nonspecific atrophy and end-stage atrophy. Autopsy findings in two patients are also described, suggesting length dependent central motor axon degeneration, peripheral motor axon degeneration, possible spinal motor neuron degeneration, and accumulation of beta amyloid precursor protein inclusions in select brainstem nuclei. Exome sequencing and homozygosity mapping identified a homozygous PRUNE1 mutation in a canonical splice site, which produces two abnormal PRUNE1 mRNA products. Based on our studies and the histopathology and phenotypic data, we provide further evidence that this disorder leads to a neurodegenerative disease affecting both the peripheral and central nervous systems and suggest that the pathogenic c.521-2A>G mutation could lead to an altered effect on tubulin dynamics.

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.

SMA valiant trial: a prospective, double-blind, placebo-controlled trial of valproic acid in ambulatory adults with spinal muscular atrophy.

INTRODUCTION: An open-label trial suggested that valproic acid (VPA) improved strength in adults with spinal muscular atrophy (SMA). We report a 12-month, double-blind, cross-over study of VPA in ambulatory SMA adults. METHODS: There were 33 subjects, aged 20­55 years, included in this investigation. After baseline assessment, subjects were randomized to receive VPA (10­20 mg/kg/day) or placebo. At 6 months, patients were switched to the other group. Assessments were performed at 3, 6, and 12 months. The primary outcome was the 6-month change in maximum voluntary isometric contraction testing with pulmonary, electrophysiological, and functional secondary outcomes. RESULTS: Thirty subjects completed the study. VPA was well tolerated, and compliance was good. There was no change in primary or secondary outcomes at 6 or 12 months. CONCLUSIONS: VPA did not improve strength or function in SMA adults. The outcomes used are feasible and reliable and can be employed in future trials in SMA adults.

A novel mutation in KIAA0196: identification of a gene involved in Ritscher-Schinzel/3C syndrome in a First Nations cohort.

BACKGROUND: Ritscher-Schinzel syndrome (RSS) is a clinically heterogeneous disorder characterised by distinctive craniofacial features in addition to cerebellar and cardiac anomalies. It has been described in different populations and is presumed to follow autosomal recessive inheritance. In an effort to identify the underlying genetic cause of RSS, affected individuals from a First Nations (FN) community in northern Manitoba, Canada, were enrolled in this study. METHODS: Homozygosity mapping by SNP array and Sanger sequencing of the candidate genes in a 1Mb interval on chromosome 8q24.13 were performed on genomic DNA from eight FN RSS patients, eight of their parents and five unaffected individuals (control subjects) from this geographic isolate. RESULTS: All eight patients were homozygous for a novel splice site mutation in KIAA0196. RNA analysis revealed an approximate eightfold reduction in the relative amount of a KIAA0196 transcript lacking exon 27. A 60% reduction in the amount of strumpellin protein was observed on western blot. CONCLUSIONS: We have identified a mutation in KIAA0196 as the cause of the form of RSS characterised in our cohort. The ubiquitous expression and highly conserved nature of strumpellin, the product of KIAA0196, is consistent with the complex and multisystem nature of this disorder.

Solving the puzzle of spinal muscular atrophy: what are the missing pieces?

Spinal muscular atrophy (SMA) is an autosomal recessive, lower motor neuron disease. Clinical heterogeneity is pervasive: three infantile (type I-III) and one adult-onset (type IV) forms are recognized. Type I SMA is the most common genetic cause of death in infancy and accounts for about 50% of all patients with SMA. Most forms of SMA are caused by mutations of the survival motor neuron (SMN1) gene. A second gene that is 99% identical to SMN1 (SMN2) is located in the same region. The only functionally relevant difference between the two genes identified to date is a C → T transition in exon 7 of SMN2, which determines an alternative spliced isoform that predominantly excludes exon 7. Thus, SMN2 genes do not produce sufficient full length SMN protein to prevent the onset of the disease. Since the identification of the causative mutation, biomedical research of SMA has progressed by leaps and bounds: from clues on the function of SMN protein, to the development of different models of the disease, to the identification of potential treatments, some of which are currently in human trials. The aim of this review is to elucidate the current state of knowledge, emphasizing how close we are to the solution of the puzzle that is SMA, and, more importantly, to highlight the missing pieces of this puzzle. Filling in these gaps in our knowledge will likely accelerate the development and delivery of efficient treatments for SMA patients and be a prerequisite towards achieving our final goal, the cure of SMA.

Generation of Conditional Knockout Alleles for PRUNE-1.

PRUNE1 is a member of the aspartic acid-histidine-histidine (DHH) protein superfamily, which could display an exopolyphosphatase activity and interact with multiple cellular proteins involved in the cytoskeletal rearrangement. It is widely expressed during embryonic development and is essential for embryogenesis. PRUNE1 could also be critical for postnatal development of the nervous system as it was found to be mutated in patients with microcephaly, brain malformations, and neurodegeneration. To determine the cellular function of PRUNE1 during development and in disease, we have generated conditional mouse alleles of the Prune1 in which loxP sites flank exon 6. Crossing these alleles with a ubiquitous Cre transgenic line resulted in a complete loss of PRUNE1 expression and embryonic defects identical to those previously described for Prune1 null embryos. In addition, breeding these alleles with a Purkinje cell-specific Cre line (Pcp2-Cre) resulted in the loss of Purkinje cells similar to that observed in patients carrying a mutation with loss of PRUNE1 function. Therefore, the Prune1 conditional mouse alleles generated in this study provide important genetic tools not only for dissecting the spatial and temporal roles of PRUNE1 during development but also for understanding the pathogenic role of PRUNE1 dysfunction in neurodegenerative or neurodevelopmental disease. In addition, from this work, we have described an approach that allows one to efficiently generate conditional mouse alleles based on mouse zygote electroporation.

Effects of 2,4-diaminoquinazoline derivatives on SMN expression and phenotype in a mouse model for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA), one of the most common genetic causes of infant death, results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of survival motor neuron (SMN) protein. In humans, the SMN gene is duplicated; SMA results from the loss of SMN1 but SMN2 remains intact. SMA severity is related to the copy number of SMN2. Compounds which increase the expression of SMN2 could, therefore, be potential therapeutics for SMA. Ultrahigh-throughput screening recently identified substituted quinazolines as potent SMN2 inducers. A series of C5-quinazoline derivatives were tested for their ability to increase SMN expression in vivo. Oral administration of three compounds (D152344, D153249 and D156844) to neonatal mice resulted in a dose-dependent increase in Smn promoter activity in the central nervous system. We then examined the effect of these compounds on the progression of disease in SMN lacking exon 7 (SMNDelta7) SMA mice. Oral administration of D156844 significantly increased the mean lifespan of SMNDelta7 SMA mice by approximately 21-30% when given prior to motor neuron loss. In summary, the C5-quinazoline derivative D156844 increases SMN expression in neonatal mouse neural tissues, delays motor neuron loss at PND11 and ameliorates the motor phenotype of SMNDelta7 SMA mice.

A newborn with spinal muscular atrophy type 0 presenting with a clinicopathological picture suggestive of myotubular myopathy.

We report a male term newborn with genetically confirmed spinal muscular atrophy type 0, presenting with arthrogryposis and severe generalized weakness and requiring ventilatory support. Muscle biopsy revealed fibers with central nuclei resembling myotubes and negative myotubularin immunohistochemical staining compared with a control muscle biopsy. The absence of myotubularin associated with survival motor neuron protein deficiency suggests that survival motor neuron protein may have a role in muscle fiber maturation and myotubularin expression. Studying the pathology of this rare and lethal neonatal form of spinal muscular atrophy may further our understanding of spinal muscular atrophy pathogenesis.

Perspectives on clinical trials in spinal muscular atrophy.

Spinal muscular atrophy is one of the most heterogeneous of the single-gene neuromuscular disorders. The broad spectrum of severity, with onset from the prenatal period to adulthood, presents unique challenges in the design and implementation of clinical trials. The clinical classification of subjects into severe (type 1), intermediate (type 2), and mild (type 3) subtypes has proved useful both in enhancing communication among clinicians internationally and in forging the collaborative development of outcome measures for clinical trials. Ideally, clinical trial design in spinal muscular atrophy must take into account the spinal muscular atrophy type, patient age, severity-of-affection status, nature of the therapeutic approach, timing of the proposed intervention relative to disease progression, and relative homogeneity of the cohort to be studied. Following is an overview of the challenges and opportunities, current and future therapeutic strategies, and progress to date in clinical trials in spinal muscular atrophy.

Characterization of the survival motor neuron (SMN) promoter provides evidence for complex combinatorial regulation in undifferentiated and differentiated P19 cells.

There exist two SMN (survival motor neuron) genes in humans, the result of a 500 kb duplication in chromosome 5q13. Deletions/mutations in the SMN1 gene are responsible for childhood spinal muscular atrophy, an autosomal recessive neurodegenerative disorder. While the SMN1 and SMN2 genes are not functionally equivalent, up-regulation of the SMN2 gene represents an important therapeutic target. Consequently, we exploited in silico, in vitro and in vivo approaches to characterize the core human and mouse promoters in undifferentiated and differentiated P19 cells. Phylogenetic comparison revealed four highly conserved regions that contained a number of cis-elements, only some of which were shown to activate/repress SMN promoter activity. Interestingly, the effect of two Sp1 cis-elements varied depending on the state of P19 cells and was only observed in combination with a neighbouring Ets cis-element. Electrophoretic mobility-shift assay and in vivo DNA footprinting provided evidence for DNA-protein interactions involving Sp, NF-IL6 and Ets cis-elements, whereas transient transfection experiments revealed complex interactions involving these recognition sites. SMN promoter activity was strongly regulated by an NF-IL6 response element and this regulation was potentiated by a downstream Ets element. In vivo results suggested that the NF-IL6 response must function either via a protein-tethered transactivation mechanism or a transcription factor binding an upstream element. Our results provide strong evidence for complex combinatorial regulation and suggest that the composition or state of the basal transcription complex binding to the SMN promoter is different between undifferentiated and differentiated P19 cells.

Protective effects of butyrate-based compounds on a mouse model for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a childhood-onset degenerative disease resulting from the selective loss of motor neurons in the spinal cord. SMA is caused by the loss of SMN1 (survival motor neuron 1) but retention of SMN2. The number of copies of SMN2 modifies disease severity in SMA patients as well as in mouse models, making SMN2 a target for therapeutics development. Sodium butyrate (BA) and its analog (4PBA) have been shown to increase SMN2 expression in SMA cultured cells. In this study, we examined the effects of BA, 4PBA as well as two BA prodrugs-glyceryl tributyrate (BA3G) and VX563-on the phenotype of SMNΔ7 SMA mice. Treatment with 4PBA, BA3G and VX563 but not BA beginning at PND04 significantly improved the lifespan and delayed disease end stage, with administration of VX563 also improving the growth rate of these mice. 4PBA and VX563 improved the motor phenotype of SMNΔ7 SMA mice and prevented spinal motor neuron loss. Interestingly, neither 4PBA nor VX563 had an effect on SMN expression in the spinal cords of treated SMNΔ7 SMA mice; however, they inhibited histone deacetylase (HDAC) activity and restored the normal phosphorylation states of Akt and glycogen synthase kinase 3ß, both of which are altered by SMN deficiency in vivo. These observations show that BA-based compounds with favorable pharmacokinetics ameliorate SMA pathology possibly by modulating HDAC and Akt signaling.

Spinal Muscular Atrophy Biomarker Measurements from Blood Samples in a Clinical Trial of Valproic Acid in Ambulatory Adults.

BACKGROUND: Clinical trials of therapies for spinal muscular atrophy (SMA) that are designed to increase the expression the SMN protein ideally include careful assessment of relevant SMN biomarkers. OBJECTIVE: In the SMA VALIANT trial, a recent double-blind placebo-controlled crossover study of valproic acid (VPA) in ambulatory adult subjects with SMA, we investigated relevant pharmacodynamic biomarkers in blood samples from SMA subjects by direct longitudinal measurement of histone acetylation and SMN mRNA and protein levels in the presence and absence of VPA treatment. METHODS: Thirty-three subjects were randomized to either VPA or placebo for the first 6 months followed by crossover to the opposite arm for an additional 6 months. Outcome measures were compared between the two treatments (VPA and placebo) using a standard crossover analysis. RESULTS: A significant increase in histone H4 acetylation was observed with VPA treatment (p = 0.005). There was insufficient evidence to suggest a treatment effect with either full length or truncated SMN mRNA transcript levels or SMN protein levels. CONCLUSIONS: These measures were consistent with the observed lack of change in the primary clinical outcome measure in the VALIANT trial. These results also highlight the added benefit of molecular and pharmacodynamic biomarker measurements in the interpretation of clinical trial outcomes.

SMA CARNIVAL TRIAL PART II: a prospective, single-armed trial of L-carnitine and valproic acid in ambulatory children with spinal muscular atrophy.

BACKGROUND: Multiple lines of evidence have suggested that valproic acid (VPA) might benefit patients with spinal muscular atrophy (SMA). The SMA CARNIVAL TRIAL was a two part prospective trial to evaluate oral VPA and L-carnitine in SMA children. Part 1 targeted non-ambulatory children ages 2-8 in a 12 month cross over design. We report here Part 2, a twelve month prospective, open-label trial of VPA and L-carnitine in ambulatory SMA children. METHODS: This study involved 33 genetically proven type 3 SMA subjects ages 3-17 years. Subjects underwent two baseline assessments over 4-6 weeks and then were placed on VPA and L-carnitine for 12 months. Assessments were performed at baseline, 3, 6 and 12 months. Primary outcomes included safety, adverse events and the change at 6 and 12 months in motor function assessed using the Modified Hammersmith Functional Motor Scale Extend (MHFMS-Extend), timed motor tests and fine motor modules. Secondary outcomes included changes in ulnar compound muscle action potential amplitudes (CMAP), handheld dynamometry, pulmonary function, and Pediatric Quality of Life Inventory scores. RESULTS: Twenty-eight subjects completed the study. VPA and carnitine were generally well tolerated. Although adverse events occurred in 85% of subjects, they were usually mild and transient. Weight gain of 20% above body weight occurred in 17% of subjects. There was no significant change in any primary outcome at six or 12 months. Some pulmonary function measures showed improvement at one year as expected with normal growth. CMAP significantly improved suggesting a modest biologic effect not clinically meaningful. CONCLUSIONS: This study, coupled with the CARNIVAL Part 1 study, indicate that VPA is not effective in improving strength or function in SMA children. The outcomes used in this study are feasible and reliable, and can be employed in future trials in SMA. TRIAL REGSITRATION: Clinicaltrials.gov NCT00227266.

SMA CARNI-VAL trial part I: double-blind, randomized, placebo-controlled trial of L-carnitine and valproic acid in spinal muscular atrophy.

BACKGROUND: Valproic acid (VPA) has demonstrated potential as a therapeutic candidate for spinal muscular atrophy (SMA) in vitro and in vivo. METHODS: Two cohorts of subjects were enrolled in the SMA CARNIVAL TRIAL, a non-ambulatory group of "sitters" (cohort 1) and an ambulatory group of "walkers" (cohort 2). Here, we present results for cohort 1: a multicenter phase II randomized double-blind intention-to-treat protocol in non-ambulatory SMA subjects 2-8 years of age. Sixty-one subjects were randomized 1:1 to placebo or treatment for the first six months; all received active treatment the subsequent six months. The primary outcome was change in the modified Hammersmith Functional Motor Scale (MHFMS) score following six months of treatment. Secondary outcomes included safety and adverse event data, and change in MHFMS score for twelve versus six months of active treatment, body composition, quantitative SMN mRNA levels, maximum ulnar CMAP amplitudes, myometry and PFT measures. RESULTS: At 6 months, there was no difference in change from the baseline MHFMS score between treatment and placebo groups (difference = 0.643, 95% CI = -1.22-2.51). Adverse events occurred in >80% of subjects and were more common in the treatment group. Excessive weight gain was the most frequent drug-related adverse event, and increased fat mass was negatively related to change in MHFMS values (p = 0.0409). Post-hoc analysis found that children ages two to three years that received 12 months treatment, when adjusted for baseline weight, had significantly improved MHFMS scores (p = 0.03) compared to those who received placebo the first six months. A linear regression analysis limited to the influence of age demonstrates young age as a significant factor in improved MHFMS scores (p = 0.007). CONCLUSIONS: This study demonstrated no benefit from six months treatment with VPA and L-carnitine in a young non-ambulatory cohort of subjects with SMA. Weight gain, age and treatment duration were significant confounding variables that should be considered in the design of future trials. TRIAL REGISTRY: Clinicaltrials.gov NCT00227266.

Adrenocortical functioning in boys with attention-deficit/hyperactivity disorder: examining subtypes of ADHD and associated comorbid conditions.

Disruptions to hypothalamic-pituitary-adrenal (HPA) axis function have been associated with varying forms of psychopathology in children. Studies suggesting children with ADHD have blunted HPA function have been complicated by the prevalence of comorbid diagnoses and heterogeneity of ADHD. The goals of this research were to assess the relations between waking and stress-response salivary cortisol levels and comorbid disruptive behavior (DBD) and anxiety (AnxD) disorders and problems in boys with ADHD, and to examine whether cortisol levels varied across ADHD subtypes. One hundred seventy elementary school-age boys with ADHD provided salivary cortisol at waking and in reaction to venipuncture. Parent reports were used to assess boys' psychiatric diagnoses and severity of behavioral problems. Boys' comorbid AnxD and anxiety problems were associated with greater cortisol reactivity, whereas boys' comorbid DBD and oppositional problems predicted diminished adrenocortical activity. Reactive cortisol increases were greatest in boys with ADHD and comorbid AnxD, but without DBD. ADHD subtypes were not differentially associated with waking, pre-stress baseline, or reactive cortisol levels. However, comorbid DBD predicted decreased cortisol reactivity in boys with inattentive and hyperactive subtypes of ADHD, but not in boys with combined subtype of ADHD. The results clarify previous patterns of distinct and divergent dysregulations of HPA function associated with boys' varying kinds of psychopathology.

Phase II open label study of valproic acid in spinal muscular atrophy.

UNLABELLED: Preliminary in vitro and in vivo studies with valproic acid (VPA) in cell lines and patients with spinal muscular atrophy (SMA) demonstrate increased expression of SMN, supporting the possibility of therapeutic benefit. We performed an open label trial of VPA in 42 subjects with SMA to assess safety and explore potential outcome measures to help guide design of future controlled clinical trials. Subjects included 2 SMA type I ages 2-3 years, 29 SMA type II ages 2-14 years and 11 type III ages 2-31 years, recruited from a natural history study. VPA was well-tolerated and without evident hepatotoxicity. Carnitine depletion was frequent and temporally associated with increased weakness in two subjects. Exploratory outcome measures included assessment of gross motor function via the modified Hammersmith Functional Motor Scale (MHFMS), electrophysiologic measures of innervation including maximum ulnar compound muscle action potential (CMAP) amplitudes and motor unit number estimation (MUNE), body composition and bone density via dual-energy X-ray absorptiometry (DEXA), and quantitative blood SMN mRNA levels. Clear decline in motor function occurred in several subjects in association with weight gain; mean fat mass increased without a corresponding increase in lean mass. We observed an increased mean score on the MHFMS scale in 27 subjects with SMA type II (p

Survival motor neuron (SMN) protein: role in neurite outgrowth and neuromuscular maturation during neuronal differentiation and development.

Childhood spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by absent or deficient full-length survival motor neuron (SMN) protein. Clinical studies and animal models suggest that SMA is a developmental defect in neuromuscular interaction; however, the role of SMN in this process remains unclear. In the present study, we have determined the subcellular localization of SMN during retinoic-acid-induced neuronal differentiation of mouse embryonal teratocarcinoma P19 cells as well as in skeletal muscle during the critical period of neuromuscular maturation. We demonstrate, for the first time, SMN accumulation in growth-cone- and filopodia-like structures in both neuronal- and glial-like cells, identifying SMN as a new growth cone marker. Indeed, SMN was present at the leading edge of neurite outgrowths, suggesting that SMN may play a role in this process. In addition, SMN was detected as small dot-like particles within the cytoplasm of skeletal muscle during the first 2 weeks after birth, but their number peaked by P6. Intense SMN staining in neuromuscular junctions was observed throughout the entire postnatal period examined. Taken together, these results suggest that SMN may indeed fulfill neuronal- and muscle-specific functions, providing a more plausible mechanism explaining motor neuron degeneration and associated denervation atrophy of skeletal muscles in SMA. The primary SMA pathology most likely initiates in the peripheral axon--the result of deficient neurite outgrowth and/or neuromuscular maturation.

Spinal muscular atrophy.

Spinal muscular atrophy is a common genetic disease of the motor neuron (frequency of eight cases per 100,000 live births) with a high mortality during infancy and no known treatment. Death is caused by severe and progressive restrictive lung disease. New information regarding the nature and function of the SMN protein and the availability of new pharmacologic agents now make it possible to consider clinical trials in this disease. Rehabilitation and proper management of medical complications have improved both the quality and duration of life for children with spinal muscular atrophy.