Pontocerebellar hypoplasia (PCH) is a clinically and genetically heterogeneous group of autosomal recessive disorders characterized by cerebellar hypoplasia or atrophy, variable pontine atrophy, and progressive microcephaly with global developmental delay7
. PCH1 is a distinctive subtype of PCH, characterized by diffuse muscle wasting secondary to spinal cord anterior horn cell loss and cerebellar hypoplasia.3–6
The diagnosis of PCH1 is often delayed or never made because the combination of cerebellar and spinal motor neuron degeneration is not commonly recognized, and the presentation of diffuse weakness and devastating brain involvement is atypical of classical proximal spinal muscular atrophy (SMA)8
. The literature contains only a handful of case series9–12
and reports on PCH113–19
. The responsible gene has not been identified in the majority of PCH1 patients. Recessive mutations have been found in vaccinia-related kinase 1 (VRK1)20
, mitochondrial arginyl-transfer RNA synthetase (RARS2)21
, and tRNA splicing endonuclease homolog 54 (TSEN54)22
in single individuals with PCH1. In PCH without SMA, TSEN54
mutations account for most cases of PCH2 and PCH423,21
, while RARS2
mutations have been found in two families with PCH624,25
We identified Family 1 in which four children were floppy at birth, with ocular motor apraxia, progressive muscle wasting, distal contractures, progressive microcephaly, growth retardation, global developmental delay, and never reached any motor milestone or spoke. Although normal in size at birth, in all four the head circumference, height, and weight dropped to below the 5th
percentile by age 7–10 months. Magnetic resonance imaging demonstrated marked cerebellar atrophy with prominent sulci and decreased volume of folia () compared to age- and gender-matched normal individuals (). In the patients, the brainstem and the cerebral cortex appear normal in configuration but are small. Electromyography showed in one patient neurogenic motor changes () exemplified by a single fast-firing (25 Hz) wave complex that was polyphasic (crossing the baseline multiple times) and unstable. The high frequency firing in the absence of other complexes suggests a loss of axons, while unstable polyphasic units are manifestations of reinnervation in response to denervation. In a younger patient we observed borderline neurogenic motor changes () with a normal recruitment pattern but occasional large-amplitude motor unit action potentials (~4.5 mV) suggestive of reinnervation, compared to normal (), which demonstrates multiple distinct wave-complexes of normal amplitudes (200–400 μV) that represent preserved motor axons without injury. Nerve conduction studies showed motor response with severely reduced amplitudes but normal sensory responses (Supplementary Table 1
). Furthermore, when the oldest child died at age 18 years after a respiratory infection, the autopsy revealed a severe loss of cerebellar () and spinal motor neurons () compared to control (). These clinical features are most consistent with PCH1.
Figure 1 Neuroimaging, neuromuscular, and pathological features in Family 1. a. Sagittal T2- and b. coronal T1-weighted images from the oldest surviving sibling who is now 18 years old demonstrate the presence of all cerebellar lobules yet with marked atrophy (more ...)
No PCH1 genes were known when this study began. We performed a genome scan, which narrowed the candidate regions to four subchromosomal loci with more than 100 candidate genes (Supplementary Fig. 1
). To identify the responsible gene, we captured the exome using SureSelect Human All Exon kit (Agilent G3362) and sequenced on a Genome Analyzer IIx (Illumina Inc.). This analysis yielded one candidate variant fulfilling the requirement of rare biallelic variants within the intervals identical by descent in all the affected individuals: EXOSC3
). We did not observe variants in VRK1, RARS2
, or TSEN54
previously reported in PCH1.
There are multiple alternative splice forms of EXOSC3
, with the longest reading frame spanning 4 exons over 5,119 bases (NM_016042.2) and encoding a 275-amino acid protein, human exosome component 3 (EXOSC3), also known as the ribosomal RNA-processing protein 40 (RRP40) (NP_057126.2). EXOSC3 is a core component of the human RNA exosome complex (distinct from exosome vesicles) present in the cytoplasm and the nucleus, especially enriched in the nucleolus26
. The N-terminal (NT) domain and putative RNA binding S1 and KH domains are evolutionarily conserved ().
Figure 2 EXOSC3 mutations in PCH1. a. Genomic structure of EXOSC3, with four exons in open boxes and mutations highlighted in magenta. circle- missense mutation; triangle- deletion mutation; star- splice site mutation. b. Alignment of orthologous sequences in (more ...)
We confirmed genotype-phenotype cosegregation in this family by Sanger sequencing. To validate the association between EXOSC3
mutations and PCH1, we sequenced all exons and flanking introns of EXOSC3
(Supplementary Table 2
) in the index patients from 12 additional PCH1 families. Eight probands had recessive mutations in the gene (; ). All available parent samples were heterozygous. None of the mutations were found in Turkish (n=94), Czech (n=96), or North American (n=189) control individuals. A more recent review of databases, including the NHLBI Exome Sequencing Project, showed that Asp132Ala has been observed in 6 of 4870 exomes, with an estimated allele frequency of 0.0012. None of the other mutations has been previously reported.
Ethnic origins and EXOSC3 mutations in subjects with PCH1. Mutations in EXOSC3 were identified by exome sequencing in affected subjects in Family 1 and further investigated in DNA samples from Families 2–13 by targeted sequencing.
The Asp132Ala mutation was present in seven of the nine mutation-positive families (; ). This mutation altered a highly conserved amino acid residue in the putative RNA binding S1 domain; the crystal structure suggests that Asp132 may be important for inter-subunit interaction within the exosome complex27
. We genotyped the probands of Families 1–3 homozygous for Asp132Ala to find identical haplotypes in a 1cM region flanking the mutation locus, suggesting an ancestral origin (Supplementary Table 3
We found three additional missense mutations. Two mutations, Gly31Ala and Trp238Arg, were present in Family 4, with parents as carriers. Gly31Ala was homozygous in the patient in Family 9. Strictly conserved from yeast to human, Gly31 in the NT domain appears to be involved in inter-subunit interaction27
, while Trp238 is in the putative RNA-binding KH domain27
. In Family 8, the patient harbored Asp132Ala in trans with another missense mutation in the S1 domain: c.415G>C; p.Ala139Pro. (; Supplementary Note
We identified one frameshift mutation: a 10-nucleotide deletion in Family 5, predicted to prematurely terminate the protein; the faulty transcript may be subject to nonsense-mediated mRNA degradation. In silico
analysis of the intronic mutation c.475-12A>G in Family 6 suggested that it may introduce a new splice site just upstream of the normal splice acceptor for exon 3. RT-PCR in expression studies demonstrated mainly skipping of exon 3 (shifting the reading frame) and evidence of aberrant splicing (which incorporated 11 nucleotides upstream of the normal splice site), with a minority of transcripts having normal splicing (Supplementary Fig. 2
& Supplementary Table 2
That biallelic missense, frameshift, and splice site mutations all led to the same clinical manifestations suggests that they may be null or hypomorphic alleles. Since all components of the exosome are essential for viability1
, it is unlikely that PCH1 patients harbor biallelic null mutations; it is more likely that the missense mutations are hypomorphic while the frameshift mutations could be null. In silico
analyses predicted detrimental consequences from the missense mutations (Supplementary Table 4
). The standard marker for impaired exosome function has long been an abnormal accumulation of unprocessed rRNA1
, which we did not observe in fibroblasts from the patients in Family 1 (Supplementary Fig. 3
), suggesting that the impact of the homozygous Asp132Ala mutations in EXOSC3 may be more nuanced and subtle than a complete elimination of exosome function.
To further examine the functional effects of the mutations, we knocked down the endogenous exosc3
expression in zebrafish embryos by exosc3
-specific antisense morpholino injection (; Supplementary Table 2
; Supplementary Fig. 4
). Zebrafish embryos injected with antisense morpholinos directed against the start codon (AUG) or the splice donor site of exon 2 (SPL) of exosc3
led to a dose-dependent phenotype with short curved spine and small brain with poor motility and even death by 3 days post fertilization (dpf), compared to embryos injected with nonspecific control morpholinos (CTL) ().
Figure 3 Knockdown of exosc3 in zebrafish embryos disrupts normal development. a. Zebrafish embryos injected with exosc3-specific antisense morpholinos AUG (directed against the start codon) or SPL (directed against the splice donor site for exon 2), compared (more ...)
The observation of shrunken or collapsed hindbrain in SPL-injected embryos prompted us to further investigate hindbrain-specific cells. Whole-mount in situ
hybridization demonstrated decreased expression of atoh1a
(marker specific for dorsal hindbrain progenitors28
) by 1 dpf in the upper rhombic lip (URL) and lower rhomic lip (LRL) in embryos injected with SPL, compared to the normal pattern of robust expression of atoh1a
in CTL-injected embryos of hindbrain progenitors in the URL and the LRL bilaterally28
(). Whole-mount in situ
hybridization further showed a lack of expression of pvalb7
, which is specific for differentiated cerebellar Purkinje neurons28
, by 3 dpf in embryos injected with SPL, compared to the normal expression in distinct clusters (highlighted by *) of differentiated Purkinje cells in embryos injected with CTL ().
The abnormal phenotype from exosc3
-specific morpholino injections was largely rescued by co-injection with wildtype zebrafish exosc3
, ; Supplementary Table 5
), suggesting that the detrimental effects of the antisense morpholinos were specific to exosc3
knockdown. Co-injection with wildtype human EXOSC3
mRNA (hWT), which shares 67% identity with the zebrafish ortholog, was less effective in the rescue. Co-injection with zebrafish or human mRNA containing the mutations was ineffective, suggesting that the mutations disrupted the normal function of EXOSC3 (; Supplementary Table 5
). Survival data of embryos 1–3 dpf are stratified and summarized in Supplementary Table 5
We have discovered disease-causing mutations in a gene encoding the exosome component EXOSC3 leading to PCH1 with combined cerebellar and spinal motor neuron degeneration of infantile onset. There is clinical heterogeneity. Affected individuals in families 1 and 3 do not present with primary hypoventilation and have survived beyond infancy and early childhood, which is exceptionally unusual for ‘classical’ PCH123,7
. Furthermore, in families 1 and 2, autopsy showed profound cerebellar atrophy and variable involvement of the pons and inferior olives, suggesting a degenerative process in addition to a developmental disorder. Additional studies will facilitate endophenotype stratification of PCH1. There is clear genetic heterogeneity in PCH1, as some patients do not harbor mutations in any known PCH1 genes.
RNA exosomes are the principal enzymes that process and degrade RNA. The bulk of the human genome is transcribed to produce an extraordinary diversity of RNAs.29
The versatility and specificity of the exosome regulate the activity and maintain the fidelity of gene expression. Although exosomes are immunogenic in some patients with polymyositis-scleroderma 30,31
or chronic myelogenous leukemia 32,33
, the findings in this report are the first to establish a pathogenic role for exosome mutations in human disease. Despite a growing effort to examine exosome function and subunit contribution, its substrates have not been fully characterized in human or lower animals, and the specific contribution of each component is incompletely understood. The discovery of naturally occurring mutations in exosome components provides a valuable opportunity to define subunit contribution to exosome function. Our findings suggest that normal function of the EXOSC3 component is essential to the survival of cerebellar and spinal motor neurons. Intriguingly, RNA dysregulation is emerging as important in the etiology of motor and cerebellar degeneration. RNA processing defects are implicated in SMN1 deficiency in SMA8
. Mutations in RNA/DNA-binding proteins34–37
and pathogenic repeat expansions generating likely toxic RNA38,39
cause amyotrophic lateral sclerosis (ALS), an adult-onset motor neuron disease. RNA gain of function from noncoding repeat expansions was recently proposed to cause combined spinocerebellar and brainstem motoneuron degeneration of late onset in SCA3640
. Dysregulation of tRNA processing underlies other subtypes of PCH24,23,21
. The elucidation of the pathomechanism underlying PCH1 may lead to new insights regarding RNA processing in the development and survival of cerebellar and spinal motor neurons.