Peggy Allred

An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: A phase 1, randomised, first-in-man study

BACKGROUND Mutations in SOD1 cause 13% of familial amyotrophic lateral sclerosis. In the SOD1 Gly93Ala rat model of amyotrophic lateral sclerosis, the antisense oligonucleotide ISIS 333611 delivered to CSF decreased SOD1 mRNA and protein concentrations in spinal cord tissue and prolonged survival. We aimed to assess the safety, tolerability, and pharmacokinetics of ISIS 333611 after intrathecal administration in patients with SOD1-related familial amyotrophic lateral sclerosis. METHODS In this randomised, placebo-controlled, phase 1 trial, we delivered ISIS 333611 by intrathecal infusion using an external pump over 11·5 h at increasing doses (0·15 mg, 0·50 mg, 1·50 mg, 3·00 mg) to four cohorts of eight patients with SOD1-positive amyotrophic lateral sclerosis (six patients assigned to ISIS 333611, two to placebo in each cohort). We did the randomisation with a web-based system, assigning patients in blocks of four. Patients and investigators were masked to treatment assignment. Participants were allowed to re-enrol in subsequent cohorts. Our primary objective was to assess the safety and tolerability of ISIS 333611. Assessments were done during infusion and over 28 days after infusion. This study was registered with Clinicaltrials.gov, number NCT01041222. FINDINGS Seven of eight (88%) patients in the placebo group versus 20 of 24 (83%) in the ISIS 333611 group had adverse events. The most common events were post-lumbar puncture syndrome (3/8 [38%] vs 8/24 [33%]), back pain (4/8 [50%] vs 4/24 [17%]), and nausea (0/8 [0%] vs 3/24 [13%]). We recorded no dose-limiting toxic effects or any safety or tolerability concerns related to ISIS 333611. No serious adverse events occurred in patients given ISIS 333611. Re-enrolment and re-treatment were also well tolerated. INTERPRETATION This trial is the first clinical study of intrathecal delivery of an antisense oligonucleotide. ISIS 333611 was well tolerated when administered as an intrathecal infusion. Antisense oligonucleotides delivered to the CNS might be a feasible treatment for neurological disorders. FUNDING The ALS Association, Muscular Dystrophy Association, Isis Pharmaceuticals.

An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis

BACKGROUND Mutations in SOD1 cause 13% of familial amyotrophic lateral sclerosis. In the SOD1 Gly93Ala rat model of amyotrophic lateral sclerosis, the antisense oligonucleotide ISIS 333611 delivered to CSF decreased SOD1 mRNA and protein concentrations in spinal cord tissue and prolonged survival. We aimed to assess the safety, tolerability, and pharmacokinetics of ISIS 333611 after intrathecal administration in patients with SOD1-related familial amyotrophic lateral sclerosis. METHODS In this randomised, placebo-controlled, phase 1 trial, we delivered ISIS 333611 by intrathecal infusion using an external pump over 11·5 h at increasing doses (0·15 mg, 0·50 mg, 1·50 mg, 3·00 mg) to four cohorts of eight patients with SOD1-positive amyotrophic lateral sclerosis (six patients assigned to ISIS 333611, two to placebo in each cohort). We did the randomisation with a web-based system, assigning patients in blocks of four. Patients and investigators were masked to treatment assignment. Participants were allowed to re-enrol in subsequent cohorts. Our primary objective was to assess the safety and tolerability of ISIS 333611. Assessments were done during infusion and over 28 days after infusion. This study was registered with Clinicaltrials.gov, number NCT01041222. FINDINGS Seven of eight (88%) patients in the placebo group versus 20 of 24 (83%) in the ISIS 333611 group had adverse events. The most common events were post-lumbar puncture syndrome (3/8 [38%] vs 8/24 [33%]), back pain (4/8 [50%] vs 4/24 [17%]), and nausea (0/8 [0%] vs 3/24 [13%]). We recorded no dose-limiting toxic effects or any safety or tolerability concerns related to ISIS 333611. No serious adverse events occurred in patients given ISIS 333611. Re-enrolment and re-treatment were also well tolerated. INTERPRETATION This trial is the first clinical study of intrathecal delivery of an antisense oligonucleotide. ISIS 333611 was well tolerated when administered as an intrathecal infusion. Antisense oligonucleotides delivered to the CNS might be a feasible treatment for neurological disorders. FUNDING The ALS Association, Muscular Dystrophy Association, Isis Pharmaceuticals.

TREM2 Variant p.R47H as a Risk Factor for Sporadic Amyotrophic Lateral Sclerosis

IMPORTANCE Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which microglia play a significant and active role. Recently, a rare missense variant (p.R47H) in the microglial activating gene TREM2 was found to increase the risk of several neurodegenerative diseases, including Alzheimer disease. Whether the p.R47H variant is a risk factor for ALS is not known. OBJECTIVES To determine whether p.R47H (rs75932628) in TREM2 is a risk factor for ALS and assess whether TREM2 expression is dysregulated in disease. DESIGN, SETTING, AND PARTICIPANTS Samples of DNA from 923 individuals with sporadic ALS and 1854 healthy control individuals self-reported as non-Hispanic white were collected from ALS clinics in the United States and genotyped for the p.R47H variant in TREM2. Clinical data were obtained on ALS participants for genotype/phenotype correlations. Expression of TREM2 was measured by quantitative polymerase chain reaction and compared in spinal cord samples from 18 autopsied patients with ALS and 12 neurologically healthy controls, as well as from wild-type and transgenic SOD1G93A mice. MAIN OUTCOMES AND MEASURES Minor allele frequency of rs75932628 and relative expression of TREM2. RESULTS The TREM2 variant p.R47H was more common in patients with ALS than in the controls and is therefore a significant risk factor for ALS (odds ratio, 2.40; 95% CI, 1.29-4.15; P = 4.1×10-3). Furthermore, TREM2 expression was increased in spinal cord samples from ALS patients and SOD1G93A mice (P = 2.8×10-4 and P = 2.8×10-9, respectively), confirming dysregulated TREM2 in disease. Expression of TREM2 in the human spinal cord was negatively correlated with survival (P = .04) but not with other phenotypic aspects of disease. CONCLUSIONS AND RELEVANCE This study demonstrates that the TREM2 p.R47H variant is a potent risk factor for sporadic ALS. To our knowledge, these findings identify the first genetic influence on neuroinflammation in ALS and highlight the TREM2 signaling pathway as a therapeutic target in ALS and other neurodegenerative diseases.

Mutations in the tail domain of DYNC1H1 cause dominant spinal muscular atrophy

Objective: To identify the gene responsible for 14q32-linked dominant spinal muscular atrophy with lower extremity predominance (SMA-LED, OMIM 158600). Methods: Target exon capture and next generation sequencing was used to analyze the 73 genes in the 14q32 linkage interval in 3 SMA-LED family members. Candidate gene sequencing in additional dominant SMA families used PCR and pooled target capture methods. Patient fibroblasts were biochemically analyzed. Results: Regional exome sequencing of all candidate genes in the 14q32 interval in the original SMA-LED family identified only one missense mutation that segregated with disease state—a mutation in the tail domain of DYNC1H1 (I584L). Sequencing of DYNC1H1 in 32 additional probands with lower extremity predominant SMA found 2 additional heterozygous tail domain mutations (K671E and Y970C), confirming that multiple different mutations in the same domain can cause a similar phenotype. Biochemical analysis of dynein purified from patient-derived fibroblasts demonstrated that the I584L mutation dominantly disrupted dynein complex stability and function. Conclusions: We demonstrate that mutations in the tail domain of the heavy chain of cytoplasmic dynein (DYNC1H1) cause spinal muscular atrophy and provide experimental evidence that a human DYNC1H1 mutation disrupts dynein complex assembly and function. DYNC1H1 mutations were recently found in a family with Charcot-Marie-Tooth disease (type 2O) and in a child with mental retardation. Both of these phenotypes show partial overlap with the spinal muscular atrophy patients described here, indicating that dynein dysfunction is associated with a range of phenotypes in humans involving neuronal development and maintenance.

Exome sequencing reveals DNAJB6 mutations in dominantly-inherited myopathy

To identify the causative gene in an autosomal dominant limb‐girdle muscular dystrophy (LGMD) with skeletal muscle vacuoles.

Amyotrophic lateral sclerosis onset is influenced by the burden of rare variants in known amyotrophic lateral sclerosis genes.

To define the genetic landscape of amyotrophic lateral sclerosis (ALS) and assess the contribution of possible oligogenic inheritance, we aimed to comprehensively sequence 17 known ALS genes in 391 ALS patients from the United States.

Lack of C9ORF72 coding mutations supports a gain of function for repeat expansions in amyotrophic lateral sclerosis

Hexanucleotide repeat expansions in C9ORF72 are a common cause of familial and apparently sporadic amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). The mechanism by which expansions cause neurodegeneration is unknown, but current evidence supports both loss-of-function and gain-of-function mechanisms. We used pooled next-generation sequencing of the C9ORF72 gene in 389 ALS patients to look for traditional loss-of-function mutations. Although rare variants were identified, none were likely to be pathogenic, suggesting that mutations other than the repeat expansion are not a common cause of ALS, and providing supportive evidence for a gain-of-function mechanism. We also show by repeat-primed PCR genotyping that the C9ORF72 expansion frequency varies by geographical region within the United States, with an unexpectedly high frequency in the Mid-West. Finally we also show evidence of somatic instability of the expansion size by Southern blot, with the largest expansions occurring in brain tissue.

Dominant spinal muscular atrophy with lower extremity predominance Linkage to 14q32

Objective: Spinal muscular atrophies (SMAs) are hereditary disorders characterized by weakness from degeneration of spinal motor neurons. Although most SMA cases with proximal weakness are recessively inherited, rare families with dominant inheritance have been reported. We aimed to clinically, pathologically, and genetically characterize a large North American family with an autosomal dominant proximal SMA. Methods: Affected family members underwent clinical and electrophysiologic evaluation. Twenty family members were genotyped on high-density genome-wide SNP arrays and linkage analysis was performed. Results: Ten affected individuals (ages 7–58 years) showed prominent quadriceps atrophy, moderate to severe weakness of quadriceps and hip abductors, and milder degrees of weakness in other leg muscles. Upper extremity strength and sensation was normal. Leg weakness was evident from early childhood and was static or very slowly progressive. Electrophysiology and muscle biopsies were consistent with chronic denervation. SNP-based linkage analysis showed a maximum 2-point lod score of 5.10 (θ = 0.00) at rs17679127 on 14q32. A disease-associated haplotype spanning from 114 cM to the 14q telomere was identified. A single recombination narrowed the minimal genomic interval to Chr14: 100,220,765–106,368,585. No segregating copy number variations were found within the disease interval. Conclusions: We describe a family with an early onset, autosomal dominant, proximal SMA with a distinctive phenotype: symptoms are limited to the legs and there is notable selectivity for the quadriceps. We demonstrate linkage to a 6.1-Mb interval on 14q32 and propose calling this disorder spinal muscular atrophy–lower extremity, dominant.

Targeted sequencing and identification of genetic variants in sporadic inclusion body myositis.

Sporadic inclusion body myositis (sIBM) has clinical, pathologic and pathomechanistic overlap with some inherited muscle and neurodegenerative disorders. In this study, DNA from 79 patients with sIBM was collected and the sequencing of 38 genes associated with hereditary inclusion body myopathy (IBM), myofibrillar myopathy, Emery-Dreifuss muscular dystrophy, distal myopathy, amyotrophic lateral sclerosis and dementia along with C9orf72 hexanucleotide repeat analysis was performed. No C9orf72 repeat expansions were identified, but; 27 rare (minor allele frequency <1%) missense coding variants in several other genes were identified. One patient carried a p.R95C missense mutation in VCP and another carried a previously reported p.I27V missense mutation in VCP. Mutations in VCP cause IBM associated with Pagets disease of the bone (PDB) and fronto-temporal dementia (IBMPFD). Neither patient had a family history of weakness or manifested other symptoms reported with VCP mutations such as PDB or dementia. In vitro analysis of these VCP variants found that they both disrupted autophagy similar to other pathogenic mutations. Although no clear genetic etiology has been implicated in sIBM pathogenesis, our study suggests that genetic evaluation in sIBM may be clinically meaningful and lend insight into its pathomechanism.

Familial ALS with extreme phenotypic variability due to the I113T SOD1 mutation

We describe a large family with amyotrophic lateral sclerosis (ALS) caused by an I113T mutation in superoxide dismuatse type 1 (SOD1). The proband developed symptoms typical for ALS at age 39 years and is still walking five years later. Marked phenotypic variability is manifested by her mother with onset of gait difficulty and decision-making problems at age 67 years and a five-year course marked by progressive mild upper motor neuron weakness, frontotemporal dementia and chorea. An aunts initial symptoms included foot numbness and an uncle with the mutation is asymptomatic. Penetrance is only 50% at age 60 years and 88% at age 80 years with an 86-year-old woman harboring the mutation and having a normal neurologic examination. This family highlights the extreme variability in age of onset, clinical manifestations, disease progression and penetrance due to the I113T SOD1 mutation.