Date Published: May 03, 2018
Author(s): David T. Burns, Sandra Donkervoort, Juliane S. Müller, Ellen Knierim, Diana Bharucha-Goebel, Eissa Ali Faqeih, Stephanie K. Bell, Abdullah Y. AlFaifi, Dorota Monies, Francisca Millan, Kyle Retterer, Sarah Dyack, Sara MacKay, Susanne Morales-Gonzalez, Michele Giunta, Benjamin Munro, Gavin Hudson, Mena Scavina, Laura Baker, Tara C. Massini, Monkol Lek, Ying Hu, Daniel Ezzo, Fowzan S. AlKuraya, Peter B. Kang, Helen Griffin, A. Reghan Foley, Markus Schuelke, Rita Horvath, Carsten G. Bönnemann.
The exosome is a conserved multi-protein complex that is essential for correct RNA processing. Recessive variants in exosome components EXOSC3, EXOSC8, and RBM7 cause various constellations of pontocerebellar hypoplasia (PCH), spinal muscular atrophy (SMA), and central nervous system demyelination. Here, we report on four unrelated affected individuals with recessive variants in EXOSC9 and the effect of the variants on the function of the RNA exosome in vitro in affected individuals’ fibroblasts and skeletal muscle and in vivo in zebrafish. The clinical presentation was severe, early-onset, progressive SMA-like motor neuronopathy, cerebellar atrophy, and in one affected individual, congenital fractures of the long bones. Three affected individuals of different ethnicity carried the homozygous c.41T>C (p.Leu14Pro) variant, whereas one affected individual was compound heterozygous for c.41T>C (p.Leu14Pro) and c.481C>T (p.Arg161∗). We detected reduced EXOSC9 in fibroblasts and skeletal muscle and observed a reduction of the whole multi-subunit exosome complex on blue-native polyacrylamide gel electrophoresis. RNA sequencing of fibroblasts and skeletal muscle detected significant >2-fold changes in genes involved in neuronal development and cerebellar and motor neuron degeneration, demonstrating the widespread effect of the variants. Morpholino oligonucleotide knockdown and CRISPR/Cas9-mediated mutagenesis of exosc9 in zebrafish recapitulated aspects of the human phenotype, as they have in other zebrafish models of exosomal disease. Specifically, portions of the cerebellum and hindbrain were absent, and motor neurons failed to develop and migrate properly. In summary, we show that variants in EXOSC9 result in a neurological syndrome combining cerebellar atrophy and spinal motoneuronopathy, thus expanding the list of human exosomopathies.
The RNA exosome is a multi-protein complex that plays a vital role in gene expression via processing and degradation of mRNA.1, 2 The exosome is composed of nine subunits (EXOSC1–EXOSC9) forming a two-layered ring and is conserved in all eukaryotes.3 EXOSC4–EXOSC9 form the core, a hexamer channel through which the RNA passes, and EXOSC1–EXOSC3 make up the cap of the exosomal ring for RNA recognition and binding.4, 5 The exosome in the nucleus processes precursor RNA and degrades precursor species, cryptic transcripts, and un-spliced RNAs,6, 7, 8, 9, 10, 11 whereas in the cytoplasm, the exosome degrades defective transcripts that have evaded nuclear degradation and AU-rich element-containing mRNAs (AREs).12 Exosomal RNA degradation proceeds in the 3′-to-5′ direction and is associated with other proteins, such as EXOSC10, for catalytic activity and the nuclear exosome targeting (NEXT) complex, which binds and delivers some specific non-coding RNAs to the exosome for degradation.13, 14, 15
We report four independent affected individuals with an early-onset progressive axonal motor neuronopathy, resulting in severe weakness and respiratory impairment, in combination with cerebellar atrophy. All affected individuals harbor autosomal-recessive variants in EXOSC9, which encodes an exosomal protein. Individual 2:II-1 presented with congenital fractures and arthrogryposis at birth and subsequent symptom progression resulting in respiratory failure and death at 15 months of age. This individual carried the heterozygous null variant in combination with the missense variant, whereas the three other affected individuals carried this missense in homozygosity; this might explain the more severe clinical presentation in individual 2:II-1. Individual 1:II-1 and individual 4:II-1 showed milder phenotypes, starting with congenital esotropia and congenital nystagmus, respectively, and poor head control in a relatively normal neonatal course. Significant developmental delay, progressive muscle weakness, oculomotor dysfunction, and coordination difficulties became evident only after 6 months of age. In particular, the lower motor neuron symptoms subsequently progressed rapidly during the second year of life: these included limited spontaneous movement of the extremities, and individual 1:II-1 also required mechanical ventilation. Individual 3:II-1 had a more pronounced neonatal presentation with muscle hypotonia, weak cry, feeding difficulties, and early-onset seizures at the age of 5 months. The phenotype of individual 3:II-1 could be partially due to another, yet to be identified, recessive genetic disorder segregating in the family, given the parental consanguinity and the history of a sister who had severe spasticity and epilepsy and passed away at age 8 years. Unfortunately, DNA from the sister of individual 3:II-1 was not available for genetic testing.