Elsevier

Neurobiology of Aging

Volume 62, February 2018, Pages 243.e1-243.e6
Neurobiology of Aging

Genetic reports abstract
Investigating CCNF mutations in a Taiwanese cohort with amyotrophic lateral sclerosis

https://doi.org/10.1016/j.neurobiolaging.2017.09.031Get rights and content

Abstract

Mutations in the cyclin F gene (CCNF) have been recently identified in a small number of patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia, and their role in patients with ALS in Taiwan remains elusive. The aim of this study was to elucidate the frequency and spectrum of CCNF mutations in a Taiwanese ALS cohort of Han Chinese origin. Mutational analyses of the CCNF gene were performed using Sanger sequencing in a cohort of 255 unrelated patients with ALS. Among these patients, the genetic diagnoses of 204 patients remained unclear after mutations in SOD1, C9ORF72, TARDBP, FUS, ATXN2, OPTN, VCP, UBQLN2, SQSTM1, PFN1, HNRNPA1, HNRNPA2B1, MATR3, CHCHD10, TUBA4A, and TKB1 had been investigated. Two novel heterozygous missense mutations in CCNF, p.S222P (c.664T>C) and p.S532R (c.1596C>T), were identified; 1 in each patient with apparently sporadic ALS. In vitro functional study demonstrated that both mutations result in a general and cyclin F-mediated ubiquitin-proteasome pathway dysfunction. The frequency of CCNF mutations in ALS patients in Taiwan is, therefore, approximately 0.8% (2/255). These findings expand the mutational spectrum of CCNF and also emphasize the pathogenic role of CCNF mutations in ALS.

Introduction

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by a rapidly progressive degeneration of motor neurons in the brain and spinal cord, resulting in progressive weakness and atrophy, bulbar palsy, and consequent respiratory failure with a short survival, typically within 3–5 years of symptom onset (Testa et al., 2004). Although the exact cause of ALS in most patients remains unclear, the genetic contributions to ALS pathogenesis have clearly demonstrated that approximately 10% of patients inherit ALS from their parents. To date, more than 50 genes have been implicated in ALS pathogenesis, and mutations in at least 10 of these genes have been unequivocally demonstrated to cause familial ALS (Peters et al., 2015, Taylor et al., 2016). Moreover, only SOD1, FUS, TARDBP, C9ORF72, VCP, and TBK1 account for a significant number of patients with ALS, indicating a high degree of genetic heterogeneity of ALS (Peters et al., 2015, Renton et al., 2014, Sreedharan and Brown, 2013). However, the roles of several newly identified causal genes of ALS, such as CCNF (Williams et al., 2016), are yet to be completely understood because of the lack of adequate relevant studies.

The CCNF gene encodes cyclin F, a protein of 786 amino acids, comprising 3 functional modules: a pseudocatalytic module containing the F-box domain and a nuclear localization signal, a substrate recruitment module containing 2 cyclin domains, and a regulatory module containing an nuclear localization signal, and a short amino acid sequence enriched in proline, glutamic acid, serine, and threonine, referred to as PEST (D'Angiolella et al., 2013). Cyclin F is the founding member of the family of F-box proteins, which are the substrate recognition subunits of Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes and mediate the ubiquitination and proteasome-dependent degradation of eukaryotic proteins (Bai et al., 1996). Cyclin F is crucial for genome stability; it regulates deoxyribonucleotide triphosphate levels (D'Angiolella et al., 2012), centrosome duplication (D'Angiolella et al., 2010), and spindle assembly (Emanuele et al., 2011) and also suppress DNA rereplication (Walter et al., 2016) by targeting and modulating the levels of the SCFcyclin F substrates, including ribonucleoside-diphosphate reductase subunit M2 (RRM2), centriolar coiled-coil protein of 110 kDa, nucleolar- and spindle-associated protein, and cell division cycle 6 protein. A recent study revealed that cyclin F interacts with B-Myb to regulate DNA damage-induced checkpoint signaling (Klein et al., 2015).

Recently, CCNF mutations were identified in patients with familial or sporadic ALS and/or frontotemporal dementia (FTD) (Williams et al., 2016). The p.S621G mutation was identified in a large family of British ancestry with ALS/FTD through linkage analysis and exome sequencing. Further screening of CCNF mutations in the ALS/FTD cohorts of different populations in Europe, North America, Japan, and Australia led to the identification of 5 and 19 different mutations in patients with familial and sporadic ALS, respectively, suggesting that CCNF mutations are present in ALS cohorts of diverse ethnicities. A replication study on 611 patients with sporadic ALS and 1424 controls demonstrated a significant enrichment of CCNF rare variants in Australian patients with sporadic ALS. Functional analyses of CCNF mutations in neuronal cells revealed abnormal ubiquitination and accumulation of polyubiquitinated proteins, indicating the pathogenic role of CCNF mutations in ALS.

To further understand the role of CCNF mutations in ALS, we screened 255 unrelated Taiwanese patients with ALS for CCNF mutations. In addition, in vitro studies were conducted to assess the functional effects of the mutant gene products.

Section snippets

Patients

Two hundred fifty-five unrelated individuals with ALS were enrolled into this study. All participants were of Han Chinese descent and recruited from the Neurology Clinics of Taipei Veterans General Hospital with the diagnosis of probable or definite ALS based on the revised EL Escorial criteria (Brooks et al., 2000). Among the 255 ALS patients, 17 have a SOD1 mutation, 12 have a TARDBP mutation, 10 have a C9ORF72 GGGGCC hexanucleotide expansion, 7 have a FUS mutation, 2 have an ATXN2

Identification of the novel CCNF mutations

Mutational analyses of CCNF in the 255 ALS patients revealed 10 missense variants, including p.V55I (c.163G>A), p.S222P (c.664T>C) (Fig. 1A), p.R406Q (c.1217G>A), c.P422L (c.1265C>T), p.P487S (c.1459C>T), p.R521H (c.1562G>A), p.L531R (c.1592T>G), p.S532R (c.1596C>G) (Fig. 1A), p.F604I (c.1810T>A), and p.R691Q (c.2072G>A). Among these variants, only p.S222P and p.S532R were not found in the 997 Taiwanese control exomes in the Taiwan Biobank database. Both variants were identified in 1 single

Discussion

In this study, we screened a Taiwanese cohort of 255 unrelated patients with ALS for mutations in the CCNF gene and identified 2 novel heterozygous missense pathogenic mutations, p.S222P and p.S532R, each in 1 patient with apparently sporadic ALS. Although 10 CCNF missense variants were originally identified within the cohort, given that CCNF-associated ALS should be a clinical rarity, 8 of them were unlikely to be disease causing for their presence in at least 1 of the 997 control exomes in

Disclosure statement

The authors have no conflicts of interest to disclose.

Acknowledgements

The authors thank the patients who participated in this study. This work was supported by the grants from Ministry of Science and Technology, Taiwan (105-2628-B-075-002-MY3), Health Promotion Administration, Ministry of Health and Welfare, Taiwan, Taipei Veterans General Hospital (V106C-032), and Taiwan Motor Neuron Disease Association. They also thank the High-throughput Genome Analysis Core Facility of National Core Facility Program for Biotechnology of Taiwan (NSC-101-2319-B-010-001) for

References (32)

  • N.F. Bence et al.

    Impairment of the ubiquitin-proteasome system by protein aggregation

    Science

    (2001)
  • C. Bettencourt et al.

    Exome sequencing uncovers hidden pathways in familial and sporadic ALS

    Nat. Neurosci.

    (2015)
  • B.R. Brooks et al.

    El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis

    Amyotroph. Lateral Scler. Other Motor Neuron Disord.

    (2000)
  • E.T. Cirulli et al.

    Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways

    Science

    (2015)
  • V. D'Angiolella et al.

    SCFCyclin F controls centrosome homeostasis and mitotic fidelity through CP110 degradation

    Nature

    (2010)
  • H.X. Deng et al.

    Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia

    Nature

    (2011)
  • View full text