Early onset and slow progression of SCA28,
a rare dominant ataxia in a large four-generation
family with a novel AFG3L2 mutation
Ulf Edener1, Janine Wo ¨llner1, Ute Hehr2, Zacharias Kohl3, Stefan Schilling4, Friedmar Kreuz5, Peter Bauer6,
Veronica Bernard1, Gabriele Gillessen-Kaesbach1and Christine Zu ¨hlke*,1
Autosomal dominantly inherited spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders
primarily affecting the cerebellum. Genetically, 26 different loci have been identified so far, although the corresponding gene
has not yet been determined for 10 of them. Recently, mutations in the ATPase family gene 3-like 2 gene were presented to
cause SCA type 28. To define the frequency of SCA28 mutations, we performed molecular genetic analyses in 140 unrelated
familial cases with ataxia. Among other variations, we found a novel missense mutation at an evolutionarily conserved amino-
acid position using a single-strand conformation polymorphism approach, followed by DNA sequencing. This amino-acid
exchange p.E700K was detected in a four-generation German family and was not observed in a survey of 400 chromosomes
from healthy control individuals.
European Journal of Human Genetics advance online publication, 31 March 2010;doi:10.1038/ejhg.2010.40
Keywords: ataxia; SCA28; AFG3L2
Spinocerebellar ataxias (SCAs) with autosomal dominant inheritance
are a clinically and genetically heterogeneous group of neurological
disorders with overlapping and highly variable phenotypes character-
ized by progressive incoordination, dysarthria and impairment of eye
movements. To date, 26 SCA loci have been identified by linkage
analysis and, for at least 16, the respective gene has been determined.
Nine SCAs are caused by expansion of tri- or pentanucleotide repeats,
seven are due to deletions, missense, nonsense or frameshift mutations
in the corresponding genes.1
In 2006, the locus for SCA type 28 (SCA28) was mapped to
18p11.22–q11.2 in a four-generation Italian family.2After 2 years,
the first set of SCA28 mutations was reported.3At the annual meeting
of the American Society of Human Genetics 2008, Cagnoli et al and Di
Bella et al presented their results regarding the ATPase family gene 3-
like 2 gene (AFG3L2) to be causative for SCA28. Overall, they have
found at least six different missense mutations in the AFG3L2 gene in
eight families. Affected individuals show slowly progressive gait and
limb ataxia, dysarthria, hyperreflexia at lower limbs, nystagmus and
ophthalmoparesis. Onset was reported to start at juvenile age (mean
age: 27 years).
The AFG3L2 gene is composed of 17 coding exons. AFG3L2, the
encoded protein, consists of 797 amino acids and exhibits different
functional domains: an AAA consensus sequence together with an
ATP/GTP-binding site, a peptidase M41 domain containing the
HEXXH motif, which is a characteristic feature of a zinc-dependent
binding domain, and an RNA-binding region.4The ubiquitously
expressed AFG3L2 is highly homologous to paraplegin, the product of
the SPG7 gene. Mutations in the SPG7 gene are responsible for an
autosomal recessive form of hereditary spastic paraplegia. Both
AFG3L2 and paraplegin are metalloproteases that are components
of the two mitochondrial AAA (m-AAA) protease isoenzymes in the
inner mitochondrial membrane. The m-AAA heterocomplex is com-
posed of both AFG3L2 and paraplegin, whereas the m-AAA homo-
complexconsists solely of AFG3L2. These proteases are known to exert
chaperon-like activity and to participate in protein quality control. In
addition, the AFG3L2 subunit functions as a processing enzyme for
paraplegin, as well as for its own maturation.5
Different mouse models confirm the ataxia-inducing role of mutated
AFG3L2, which primarily affects neuronal tissues. Afg3l2?/?genetic
mutants lead to a severe ataxic phenotype with early lethality. In
contrast, paraplegin-deficient mice show a mild form accompanied
by late-onset axon degeneration.6Furthermore, Spg7?/?mutant mice
with a heterozygous mutation in the Afg3l2 gene are reported to be
afflicted with an ataxic phenotype that is due to an accelerated
axonopathy.7Altogether, these data indicate the important role of
AFG3L2 in neuronal function and a rather modulatory role of
A recently described mouse model, haploinsufficient for Afg3l2,
exhibits several features common to the human SCA28 phenotype.
Hence, this mouse model seems to be adequate to unravel the
pathological cascade leading to this form of SCA.8
Notably, the six AFG3L2 mutations known so far occur in exons
15 and 16, both of which contribute to the peptidase M41 domain.
Received 10 November 2009; revised 13 January 2010; accepted 25 February 2010
1Institut fu ¨r Humangenetik, Universita ¨t zu Lu ¨beck, Lu ¨beck, Germany;2Zentrum und Institut fu ¨r Humangenetik, Universita ¨tsklinikum Regensburg, Regensburg, Germany;
3Abteilung fu ¨r Molekulare Neurologie, Universita ¨tsklinikum Erlangen, Erlangen, Germany;4Klinik und Poliklinik fu ¨r Neurologie, Universita ¨tsklinikum Regensburg, Regensburg,
Germany;5Gemeinschaftspraxis fu ¨r Humangenetik, Dresden, Germany;6Institut fu ¨r Humangenetik, Universita ¨t Tu ¨bingen, Tu ¨bingen, Germany
*Correspondence: Dr C Zu ¨hlke, Institute for Human Genetics, University of Luebeck, Ratzeburger Allee 160, Luebeck D-23538, Germany. Tel: +49 451 500 2622;
Fax: +49 451 500 4187; E-mail: Christine.Zuehlke@uk-sh.de
European Journal of Human Genetics (2010), 1–4
& 2010 Macmillan Publishers Limited All rights reserved 1018-4813/10 $32.00
All these missense exchanges hit evolutionarily conserved amino acids
and none of themwas found in 200 control chromosomes. To evaluate
the frequency of AFG3L2 mutations among German ataxia cases, we
screened a group of 140 unrelated patients with a familial history of
dominant ataxia using a single-strand conformation polymorphism
(SSCP) approach. With respect to the clustering of the described
missense mutations, only the affected exons 15 and 16 were analysed.
In addition, to check the SSCP results and to reveal intragenic
variations, all 17 coding exons of the AFG3L2 gene were sequenced
for 20 index patients.
PATIENTS AND METHODS
A total of 140 unrelated German patients with familial ataxia and 200 healthy
controls were studied. Before genetic analysis, mutations in the known SCA
genes were excluded for the patients. After having obtained informed consent,
genomic DNA was extracted from peripheral blood leukocytes by standard
PCR products of exons 15 and 16 were denatured at 951C for 5min and
immediately chilled on ice. The DNA strands were electrophoretically separated
in a denaturing polyacrylamide gel (8% acrylamide and 10% urea) at 30W for
4 hours at 251C. Subsequently, the gels were silver stained.
All 17 exons of the AFG3L2 gene were amplified by PCR. Fragment
lengths were checked by electrophoretic separation on a 1.5% agarose gel.
PCR products including exons as well as flanking intronic sequences
were sequenced on both strands. Finally, sequences were analysed manually
DNA samples from 200 unrelated control individuals of German descent were
tested for the c.2098G4A exchange (p.E700K) by allele-specific PCR. Mutated
and wild-type fragments were separated on a 2.5% agarose gel for 30min.
The primer sequences used were mut-forward 5¢-ctgcaagattgatagatgata-3¢,
ENSG00000141385, AFG3L2 Homo sapiens transcript ENST00000269143,
ENSMUSP00000025408, Rattus norvegicus ENSRNOP00000024632, Canis
familiaris ENSCAFP00000027762, Pongo pygmaeus ENSPPYP00000010085,
Pan troglodytes ENSPTRP00000016808, Equus caballus ENSECAP00000004939
and Bos taurus ENSBTAP00000030993. Multiple sequence alignment was
performed by using the online programme ‘CLUSTALW’ (http://align.
genome.jp/). The clinical impact of the missense mutation p.E700K
was assessed by the online version of ‘Mutation T@ster’ (http://www.
codes(Ensembl release 54). AFG3L2Homo sapiens
The first clinical signs in the male patient IV4were noted at the age of 66
and included impaired fine and gross motor skills and gait instability. MR
imaging at that time confirmed cerebellar hypoplasia. Currently, at the age of
12years, he attends elementary school and has problems with working speed,
progredient difficulties in writing, as well as in fine motor skills. His neuro-
logical examination revealed ataxia, dysdiadochokinesia, muscular hypotonia
and increased muscle reflexes. His 38-year-old mother, III9, reported first gait
instability and clumsiness at the age of 10 years. When the patient was re-
evaluated at the age of 36 years, she exhibited limb and gait ataxia, moderate
dysarthria and bilateral gaze-evoked nystagmus, with normal reflexes and a lack
of pyramidal signs. Further imaging studies (MRI) indicated cerebellar atrophy
without any change in supratentorial compartments (including the corpus
callosum) or in the brainstem. Despite clinical manifestation during infancy,
the phenotype that is more than 25 years after onset is remarkably mild,
allowing the patient to be completely independent.
The 44-year-old cousin III3first noted balance and writing disturbances after
delivering her twin boys at the age of 28 years. She still works part time as an
office employee, has mild dysarthria and currently needs to pay more attention
during walking and talking. Re-evaluation at the age of 38 years revealed mild
ataxia, dysdiadochokinesia, dysarthria, slight muscular hypotonia and increased
muscle reflexes. Clinical findings are summarized in Table 1.
Table 1 Clinical findings
Patient III, 9IV, 4III, 3 IV, 1 IV, 2
Age at onset
Age at exam
Normal/febrile seizures at 3 years
Normal/febrile seizures at 3 years
U Edener et al
European Journal of Human Genetics
RESULTS AND DISCUSSION
In this study, DNA samples of 140 unrelated patients with a familial
history of dominant ataxia were screened for sequence abnormalities
in exons 15 and 16 of the AFG3L2 gene using an SSCP approach.
Furthermore, to check the SSCP results and to reveal intragenic DNA
sequence variations, all 17 coding exons were sequenced for 20 index
patients. Altogether, we found five different single-nucleotide
exchanges in the coding sequence, as well as in the 3¢-UTR and
intronic regions. One of these variations leads to a change at the
amino-acid level (Table 2).
SSCP screening of 140 unrelated ataxia patients showed a peculiar-
ity in exon 16 for one sample. Sequencing of this exon revealed the
heterozygous transition c.2098G4A in a 44-year-old female patient
from Germany (III3). At the amino-acid level, this single-nucleotide
substitution results in the missense exchange p.E700K.
Remarkably, the variation c.2098G4A was not found in DNA
samples of 200 unrelated control individuals of German descent by
p.E700K segregated with the disease in four additional affected
members of the four-generation family (Figure 1). In patient IV4,
his mother III9and in the twins IV1and IV2, the first cerebellar signs
of the disease developed within the first decade of life, whereas in five
additional family members, symptoms were retrospectively noted
within the third decade of life with an unusually slow progression
and maintained free walking into the seventh decade of life. A more
severe clinical presentation was only observed in both affected mono-
zygotic twins, who in addition show a global developmental delay
and currently attend a special school for children with cognitive
impairment. However, given the normal cognitive function of the
other affected relatives in this family and the few cases described so far
in the literature, reported premature delivery and serious postnatal
adaptation problems need to be considered as important cofactors for
the more complex and severe clinical presentation of these mono-
In contrast, predictive testing of one not affected family member
revealed the wild-type sequence. On the basis of these findings, a
pathogenic impact of the missense mutation p.E700K on the ataxic
phenotype is highly presumable. This assumption is supported by the
strong conservation of glutamic acid (E) at position 700 in M.
musculus, R. norvegicus, C. familiaris, P. pygmaeus, P. troglodytes, E.
caballus and B. taurus. Furthermore, the online programme ‘Mutation
T@ster’ (http://www.mutationtaster.org/) classifies this amino-acid
exchange as being presumably disease causing.
It is noteworthy that a pathogenic missense exchange E–K is already
described in the literature.3However, the authors do not specify the
SCA28 gene name nor the exact position of this amino-acid substitution.
Thus, it remains unclear whether both missense mutations are identical.
Subsequent to the SSCP screening, exons 15 and 16 were sequenced
for 19 randomly selected individuals out of the 140 ataxia patients, as
well as for the 44-year-old female patient III3carrying the missense
mutation p.E700K. No further DNA sequence abnormality was
identified in any of the corresponding samples, indicating good
reliability of the SSCP assay. Moreover, all remaining coding exons
of the AFG3L2 gene were investigated by DNA sequence analysis for
these 20 ataxia patients in order to reveal rare and underlying
intragenic variations. This is due to the crucial necessity of gaining
Table 2 Sequence variations within the AFG3L2 gene
Localization DNA variationMissense variation Silent exchangeSNPa
Number of patients Number of controls
adbSNP identification number.
b10 out of the 18 patients’ samples are homozygous for the IVS7+6T allele.
c10 out of the 18 patients’ samples are homozygous for the c.1389A allele.
d10 out of the 18 patients’ samples are homozygous for the c.1650G allele.
e2 out of the 10 patients’ samples are homozygous for the c.*28C allele.
3679 1011 12 1314
Figure 1 Pedigree of a four-generation German family with autosomal dominant spinocerebellar ataxia. Filled symbols indicate affected subjects. Open
symbols specify unaffected spouses and presently asymptomatic family members. Deceased individuals are marked by a diagonal line. DNA samples of
subjects III3, III9, IV1, IV2and IV4were available for molecular genetic analysis (arrows).
U Edener et al
European Journal of Human Genetics
knowledge about the clinical impact of AFG3L2 variants to provide a Download full-text
firm basis for genetic testing in SCA28.
c.*28G4C were detected in intron 7 and in the 3¢-UTR of
the AFG3L2 gene, respectively. In addition to these noncoding SNPs,
the two silent exchanges, p.L463 (exon 11) and p.E550 (exon 13), were
found (Table 2). The intronic SNP and the two silent exchanges occur
exclusively in the same individuals. Primarily, a pathogenic effect
through impaired splicing would be possible for the IVS7+6C4T
exchange because of its proximity to the exon/intron boundary. However,
given the relatively high frequency of these four SNPs and the fact that
no typical splice site is generated in all cases, a disease-causing impact
through aberrant splicing is very unlikely for these variations.
Overall, only one ataxia-inducing mutation was found in 140
patients, illustrating that SCA28 is a relatively rare cause of disease
in the German population. The neurological examination of the five
affected family members further supports previous data regarding the
clinical presentation of most SCA28 patients: an early-onset cerebellar
ataxia with a slowly progressive phenotype. Notably, all AFG3L2
mutations known so far are located in a small interval between
amino acids 654 and 700 in exons 15 and 16. Hence, for diagnostic
purposes, investigating only these two exons should be taken into
account. The clustering in the AFG3L2 peptidase M41 domain, as well
as the missense nature of all described mutations, argues against
haploinsufficiency as the pathogenetic mechanism. In fact, a domi-
nant-negative effect through gain of function is more presumable.
(SNPs) IVS7+6C4T and
CONFLICT OF INTEREST
The authors declare no conflict of interest.
We thank all patients for supplying blood samples for scientific research
and their clinicians for collecting them. A part of this work was supported
by the Deutsche Forschungsgemeinschaft (DFG: ZU 136/1-2) and by the
German Heredo-Ataxia Society (DHAG).
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