Introduction

Autosomal dominant spinocerebellar ataxias (SCA) are a large heterogeneous group of neurological disorders characterized by gait and limb ataxia, dysarthria, and nystagmus in combination with other clinical signs and symptoms such as dystonia, chorea, neuropathy, and cognitive impairment. MRI typically reveals atrophy of the cerebellum. To date, 31 different SCA loci and 24 genes are known (Bird 2013). Among those, the SCA28 locus has been mapped to chromosome 18p11.22-q11.2 in a Southern Italian family (Cagnoli et al. 2006). The disease-causing gene was shown to be ATPase family gene 3-like 2 (AFG3L2) by the detection of missense mutations in patients (Mariotti et al. 2008; Di Bella et al. 2010; Cagnoli et al. 2010; Edener et al. 2010). AFG3L2 is composed of 17 exons, coding for a protein of 797 amino acids (aa). Important domains of AFG3L2 include an ATPase associated with a variety of cellular activities (AAA) and a carboxyterminal peptidase domain (M41). The AAA domain contains an ATP/GTP binding site, and the peptidase M41 domain includes a HEXXH motif characteristic of zinc binding and an RNA binding region (Banfi et al. 1999). AFG3L2 is a homo- or hetero-oligomeric ATP-dependent metalloprotease protein complex of the inner mitochondrial membrane. The hetero-oligomeric protease complex is composed of AFG3L2 and the homologous gene product of SPG7, paraplegin, both of which share 49 % of identical aa residues (Banfi et al. 1999). Loss-of-function mutations in SPG7 were found in autosomal recessive spastic paraplegia (Casari et al. 1998).

To date, 12 missense mutations affecting nine codons of AFG3L2 have been identified in single families of Caucasian origin (Mariotti et al. 2008; Di Bella et al. 2010; Cagnoli et al. 2010; Edener et al. 2010). All known mutations except one are located in exons 15 and 16 of AFG3L2. Due to the low number of reported cases, genotype–phenotype correlations are not yet possible. In most SCA28 patients, reported disease onset was during early adulthood. Progression of signs and symptoms was slow and included gait ataxia, dysarthria, increased deep tendon reflexes, and impairment of eye movements. In addition, ptosis was observed in some SCA28 patients. Most of the reported patients remain ambulatory well into their sixties (Brussino et al. 2013).

To further elucidate the genetic spectrum of AFG3L2 mutations, we investigated 28 unrelated ataxia patients of German origin with a proven family history of an autosomal dominant movement disorder. SCA loci 1–3, 6–8, 10, 12–14, 17, and 27 had been excluded previously.

Materials and Methods

Genetic Analysis

Twenty eight unrelated clinically proven SCA patients of German origin were studied. All had a positive family history consistent with autosomal dominant inheritance. MRI had revealed cerebellar atrophy in all cases. Repeat expansions in SCA1–3, 6–8, 10, 12, and 17 and point mutations in SCA13 (KCN3), SCA14 (PRKCG), and SCA27 (FGF14) had been previously excluded. All 17 exons and flanking intronic boundaries of the AFG3L2 gene (ENSG00000141385) were amplified by PCR and subsequently sequenced using the BigDye Terminator Kit v2 (Applied Biosystems) according to the supplier's instructions. SeqScape v2.5 software (Applied Biosystems) was used for data analysis. Blood samples from the 28 ataxia patients had been submitted to our laboratory by neurologists in accordance with German law for genetic testing.

Case Report

The patient came to clinical attention at age 60 due to mild gait and limb ataxia and impaired postural stability in combination with moderate dysarthria and occasional dysphagia. He displayed mild dysdiadochokinesia, dysmetria, and moderate hyperreflexia. Babinski sign was negative. Slow saccades were noted. There was no obvious cognitive impairment. The patient's feet were highly arched, and he had pallanesthesia at the first metatarsophalangeal joints. The Scale for Assessment and Rating of Ataxia (SARA) score (Schmitz-Hübsch et al. 2006) was 14.5. By history, the patient first manifested gait disturbances at age 43. Gait anomalies progressed slowly. During the last years, he fell frequently. He could not climb the stairs without aid, and he was unable to ride a bicycle. Fine motor movements were impaired. His condition worsened through fatigue and agitation. He reported occasional mild dysphagia. Family history revealed a similar movement disorder in his deceased father and in his paternal grandmother (Fig. 1a).

Fig. 1
figure 1

a Pedigree of the German SCA28 family. Black symbols indicate affected probands. The index patient is marked by an arrow. Asterisks mark two siblings of unknown gender; information about health status was not available. b DNA sequence chromatograms showing the heterozygous C to T exchange (c.2065T>C) in the index patient compared to a control. Corresponding amino acid sequence is given. c Structure of the AFG3L2 gene and composition of the deduced protein. Mutated codons are marked by asterisks. Known aa exchanges are given in black, and novel one in red. Domains are given in accordance to the Pfam database (http://pfam.sanger.ac.uk; Punta et al. 2012). MTS mitochondrial targeting sequence; TM1 and TM2 transmembrane domains 1 and 2, respectively; FtsH filamentation temperature sensitive H proteolytic domain. d Amino acid sequence alignment of parts of the M41 domain in AFG3L2 orthologs. Protein identifier numbers are given on the right. Mutation at highly conserved tyrosine residue at position 689 is highlighted in red. Known affected aa are marked by dots. Nonconserved residues are given in green. G. Gorilla; P. Pongo; C. Canis; B. Bos; R. Rattus; M. Mus; G. Gallus; X. Xenopus; D. Drosophila; C. Caenorhabditis

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Results and Discussion

Sequencing of the patient's AFG3L2 transcript (ENST00000269143) revealed a C to T transition in exon 16 at nucleotide position 2065 (c.2065T>C, cDNA.2297T>C, g.39864T>C; Fig. 1b). The nucleotide change results in the substitution of tyrosine by histidine (p.Y689H) in the M41 peptidase domain of AFG3L2 (Fig. 1c). Several reasons suggest that the nucleotide change is pathogenic:

  1. 1.

    The c.2065T>C change was not detected in the 1000 Genomes Database (http://www.1000genomes.org/), dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/), and the Exome Variant Server (http://evs.gs.washington.edu/EVS/).

  2. 2.

    Additionally, c.2065T>C was not found in 240 healthy age-matched controls of German origin (480 chromosomes).

  3. 3.

    A computer-based analysis approach using the SIFT algorithm (http://blocks.fhcrc.org/sift/SIFT.html) (Kumar et al. 2009) and the MutationTaster tool (lhttp://mutationtaster.org) predicts that the p.Y689H change is disease causing.

  4. 4.

    Tyrosine at position 689 of the M41 domain has been highly conserved during evolution. It is identical in all vertebrates studied and in invertebrates such as Caenorhabditis elegans as well (Fig. 1d).

Interestingly, of 52 amino acids of the carboxyterminal M41 domain, only 24 have been evolutionarily conserved (Fig. 1d) and seven of them were found mutated in SCA28 (Table 1) (Di Bella et al. 2010; Cagnoli et al. 2010; Edener et al. 2010). The mutation described here (p.Y689H) affects another highly conserved amino acid in this part of the M41 domain.

Table 1 AFG3L2 mutations in SCA28 patients
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To date, all mutations in SCA28 but one (c.1295A>C) in exon 10 (Di Bella et al. 2010) are located in exons 15 and 16 (Fig. 1c), respectively, that code for the M41 peptidase domain. The mutations interfere with homo- and hetero-oligomer formation of AFG3L2 (Cagnoli et al. 2010). Impairment of formation of the AFG3L2 oligomer complex affects mitochondrial protein synthesis and ribosome assembly and is the likely molecular pathogenic mechanism in SCA28 (Almajan et al. 2012).

The AFG3L2 mutation was identified in one of 28 patients in whom autosomal dominant SCAs caused by repeat expansions had been excluded. This suggests a prevalence of 3.6 % among this group of ataxia patients in Germany. This is comparable to a reported prevalence of 3.0 % in Caucasian similarly preselected patients (Cagnoli et al. 2010).

Age at onset varies widely in SCA28. While onset is during early adulthood in most cases (Cagnoli et al. 2006, 2010), childhood onset has been described in some (Edener et al. 2010). In the four-generation family described by Edener, age of disease onset varied greatly. In contrast, disease onset was comparable in the patients of the three-generation family reported here. Furthermore, while tendon reflexes were increased only moderately in the present patient, they appear to be greatly increased in most SCA28 cases (Cagnoli et al. 2006, 2010; Edener et al. 2010).

In conclusion, the findings reported support the importance of malfunction of the M41 domain of AFG3L2 in the pathogenesis of SCA28 and further document that phenotypic variability is moderate in this disorder.