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Relationship between C9orf72 repeat size and clinical phenotype
1,2,3,4 1,2 1,2
Sara Van Mossevelde , Julie van der Zee , Marc Cruts
1,2
and Christine Van Broeckhoven
Patient carriers of a C9orf72 repeat expansion exhibit Patients carrying a C9orf72 repeat expansion are remark-
remarkable heterogeneous clinical and pathological able heterogeneous in clinical presentation, not only
characteristics suggesting the presence of modifying factors. between families but also within families [3,4 ]. The
In accordance with other repeat expansion diseases, repeat majority of the expansion carriers present clinically with
length is the prime candidate as a genetic modifier. FTD and/or ALS. 73–100% of C9orf72 FTD patients
Observations of earlier onset ages in younger generations of exhibit the behavioral variant (bvFTD) [5–15]. In C9orf72
large families suggested a mechanism of disease anticipation. ALS patients, the relative frequency of a bulbar symptom
Yet, studies of repeat size and onset age have led to conflicting onset (29–89% [5,9,11,13,15–18]) is higher than in ALS
results. Also, the correlation between repeat size and diagnosis patients without the expansion [13,16–18]. Apart from
is poorly understood. We review what has been published FTD and ALS, several other clinical diagnoses have been
regarding C9orf72 repeat size as modifier for phenotypic described (Figure 1) [9,19–31]. Parkinsonism is fre-
characteristics. Conclusive evidence is lacking, partly due to quently reported in C9orf72 repeat expansion carriers,
the difficulties in accurately defining the exact repeat size and but the expansion does not seem to be associated with
the presence of repeat variability due to somatic mosaicism. Parkinson’s disease (PD) and is only rarely identified in
patients with a clinical typical or atypical PD diagnosis Addresses [15,25–27,32–36].
1
Center for Molecular Neurology, VIB, Universiteitsplein 1,
2610 Antwerp, Belgium
2 Pathologically, affected regions in the brain and spinal
Laboratory of Neurogenetics, Institute Born-Bunge, University of
C9orf72
Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium cord of repeat expansion carriers display accu-
3
Department of Neurology and Memory Clinic, Hospital Network mulation of TAR DNA binding protein(TDP)-43 pathol-
Antwerp Hoge Beuken, Commandant Weynsstraat 165, 2660 Hoboken,
ogy, mostly type B [11,14], although type A also occurs
Belgium
4 [3,5,9,16] (Figure 1). Within one single family, different
Department of Neurology, Antwerp University Hospital, Wilrijkstraat 10,
TDP subtypes can occur [3]. Although most clinical AD
2650 Edegem, Belgium
patients with a C9orf72 repeat expansion turn out to be
Corresponding author: Van Broeckhoven, Christine FTLD-TDP on neuropathology [7,19], neuropathologi-
cally confirmed AD cases have been described in expan-
sion carriers [22]. Furthermore, in one expansion carrier
Current Opinion in Genetics & Development 2017, 44:117–124 the disease was neuropathologically characterized as a
tauopathy consistent with corticobasal degeneration
This review comes from a themed issue on Molecular and genetic
pathology without any TDP-43 pathological changes in
bases of disease
the hippocampus or cerebral cortex [9]. Other neuropath-
Edited by Edward Lee and Wilma Wasco
ological characteristics in C9orf72 repeat expansion car-
riers are the presence of RNA foci and dipeptide repeat
protein pathology [58–60].
http://dx.doi.org/10.1016/j.gde.2017.02.008
0959-437X/ã 2017 The Authors. Published by Elsevier Ltd. This is an Apart from the heterogeneity in clinical and pathological
open access article under the CC BY-NC-ND license (http://creative- diagnoses, there is also a remarkable variability in onset
commons.org/licenses/by-nc-nd/4.0/).
age. Onset in C9orf72 repeat expansion carriers is
reported to range from 29 to 82 years of age [4 ,10–
12,14,16,29] with a variability up to 22 years in a single
Introduction family [3].
A pathogenic expansion of the C9orf72 repeat is the most
This marked phenotypical heterogeneity suggests the
common genetic cause of frontotemporal lobar degener-
presence of genetic modifying factors in C9orf72-related
ation (FTLD) and amyotrophic lateral sclerosis (ALS)
disease. In accordance with other repeat expansion dis-
(Figure 1). The C9orf72 repeat expansion explains 25% of
orders, the most obvious candidate to explain the variable
familial frontotemporal dementia (FTD), 37% of familial
disease expression is the size of the G C expanded
ALS and up to 88% of familial patients with both FTD 4 2
repeat.
and ALS [1,2].
www.sciencedirect.com Current Opinion in Genetics & Development 2017, 44:117–124
118 Molecular and genetic bases of disease
Figure 1
ID S SCA AD CBS PSP HD phenocopy OPCD
bvFTD phenocopy PMA C9orf72
FTLD ALS
clinical clinical FTD pathological FTLD pathological electrophysiological neuronal loss prominent neuronal loss UMN signs + LMN signs - anterior nerve roots in frontal and / or temporal lobes bvFTD PPA - spasticity - weakness - lower cranial nuclei brainstem prominent prominent - hyperactive reflexes - atrophy - Betz cells motor cortex behavioral / executive language deficits - Babinski sign -fasciculations deficits onset PNFA SD LPA TDP-43 Ta U TDP-43 Type A Type B 4R bulbar spinal CBD - dysarthria - progressive limb weakness - dysphagia - fasciculations limb muscles - tongue fasciculations - hyperreflexia limbs
Current Opinion in Genetics & Development
C9orf72 disease heterogeneity.
Most C9orf72 repeat expansion carriers exhibit frontotemporal lobar degeneration (FTLD) and/or amyotrophic lateral sclerosis (ALS). FTLD
presents clinically as frontotemporal dementia (FTD) and has two variants: the behavioral variant of FTD (bvFTD) [56] and primary progressive
aphasia (PPA) [57]. This last variant can be further separated into progressive non-fluent aphasia (PNFA), semantic dementia (SD) and logopenic
progressive aphasia (LPA), although the latter is frequently associated with underlying atypical Alzheimer’s disease pathology. ALS is the result of
a combination of upper motor neuron (UMN) and lower motor neuron (LMN) degeneration. Histopathological, most FTLD and ALS cases due to a
C9orf72 repeat expansion are characterized by TAR DNA binding protein-43 (TDP-43) inclusions, although tau-inclusions have also been reported.
Apart from ALS and FTLD, several other diagnoses have been described in expansion carriers: Alzheimer’s disease (AD), Huntington’s disease
(HD) phenocopy (not carrying a CAG repeat expansion in the HTT gene), progressive supranuclear palsy (PSP), corticobasal syndrome/
degeneration (CBS/CBD), bvFTD phenocopy syndrome (a slowly progressive bvFTD-like phenotype), primary muscular atrophy (PMA), sporadic
spinocerebellar ataxia (S SCA), olivopontocerebellar degeneration (OPCD) and intellectual disability (ID).
Normal C9orf72 repeat length smaller repeats (Figure 2). Repeat lengths between 24 and
The cut-off to discriminate between ‘normal’ repeat 28 units have been observed in ALS patients [17,18,39],
alleles and pathogenic expanded repeats is not very well while repeat alleles as short as 20 to 22 repeats have been
established. In most healthy control cohorts, normal observed in FTD patients and their affected siblings [37].
repeat lengths up to 24 units were identified [10,11,14, Co-segregation with disease of expansions as short as
17,22,29,32,37–40] (Figure 2). However, two authors 47 units and of expansions between 35 and 100 units have
reported slightly larger maximal repeat sizes of 32 and been observed in an FTD/ALS family [46 ] and two AD
35 units, respectively [6,18], and others even observed families respectively [20]. A point of discussion here is
large expansions of more than 400 units in unaffected whether these ‘intermediate’ repeat sizes in blood are
individuals, probably due to a reduced C9orf72 disease directly associated with pathology, or whether they are
penetrance and high variability in onset age [23] associated with fully expanded repeats in disease-relevant
(Figure 2). tissue due to increased instability.
In patients, the size of most repeat expansions is estimated Effect C9orf72 repeat length on clinical
to range between several hundred and several thousand phenotype
repeat units [8,23,29,39,41 ,42–44,45 ]. Although most Some authors observed significantly larger pathogenic
authors use an arbitrary cut-off of 30 or 60 units (depending expansion sizes in ALS patients than in FTD patients
on the upper limit of the repeat-primed PCR detection [47 ,48 ] (Figure 2). However, several Southern blot
method), several affected individuals were reported with hybridization studies using different measures of repeat
Current Opinion in Genetics & Development 2017, 44:117–124 www.sciencedirect.com
Relationship between C9orf72 repeat size and clinical phenotype Van Mossevelde et al. 119
Figure 2
1a 2 3 4 5 6 7 8 9 10 11 C9orf72
GGGGCC n 1 72430 60 80 400 4400
1. increased risk of ALS in comparison with FTD? increased risk of: 2. increase in age at onset? sporadic ALS? ⊕positive correlation between onset age and repeat size
3. Decrease in age at onset? PD / ET? (1) (2)
⊕ arguments for the occurrence of anticipation
OA 6 Y 6 U * clinical (1) : 3 5 most healthy individuals 0.09%* earlier onset ages in younger generations
* genetic and molecular (2) : OA 54 Y >1100U longer repeat size 0.46%* in younger generations increased CpG methylation* OA 45 Y ⊕ inverse correlation between onset age and repeat size
Current Opinion in Genetics & Development
Association between repeat length and phenotype characteristics.
On top of this figure the C9orf72 gene, consisting of 11 exons, is pictured. In the promotor region of exon 1, the G4C2 hexanucleotide repeat is
located. The letter ‘n’ indicates the number of G4C2 units. Underneath, a continuous line indicates the possible number of units (U), ranging from
1 to 4400. ‘Cut-off’ values frequently reported in literature are indicated. The color of the line indicates the estimated probability that a repeat
expansion leads to disease, ranging from bright green (least likely to lead to disease) to bright red (highest likely to lead to disease). Red crosses
indicate numbers of repeat units below 80 units that are reported in affected individuals [17,18,20,37,39,46 ], green crosses indicate numbers of
repeat units larger than 30 units that are reported in healthy individuals [6,18,23]. Most healthy individuals have repeat sizes below 24 units.
Intermediate repeat sizes between 24 and 30 units were suggested by some authors as a potential risk factor for sporadic amyotrophic lateral
sclerosis (ALS) [50 ] and Parkinson’s disease (PD) or essential tremor (ET) [15,32,34,35]. Within the pathogenic repeat size region, some authors
suggested that larger repeat sizes are more likely to lead to ALS instead of FTD [47 ,48 ]. Further, arguments for an increase in onset age (OA) in
association with an increase in repeat length has been provided by some [23,41 ,44,45 ,49 ], while others provided arguments for a decrease in
onset age in association with an increase in repeat length [46 ]. The latter is supported by evidence for the occurrence of anticipation [3,4 ,5,11–
13,15,16,46 ]. The pictured pedigrees illustrate clinical, genetic and molecular arguments for the occurrence of anticipation. Clinical affected
individuals are pictured as grey squares, unaffected individuals as white squares. The first pictured pedigree (1) shows a lower onset age in
patients of later born generations, which is a clinical argument for the occurrence of anticipation. The second pedigree (2) shows an affected
parent who carries a C9orf72 repeat expansion of only 56 units while his (still) unaffected child carries a large C9orf72 repeat expansion of more
than 1100 units, in accordance with genetic anticipation. Furthermore, the CpG methylation degree is higher in the child (0.46%) than in the parent (0.09%).
size in blood or brain could not reveal a significant developing sporadic ALS [50 ] (Figure 2). In AD, the
correlation between repeat length and clinical or patho- number of unexpanded repeats does not appear to be a
logical diagnosis [44,49 ], not even when also accounting risk factor for development of the disease [20–22] nor a
for other neurodegenerative phenotype presentations modifying factor for cerebrospinal fluid biomarkers for
besides FTD or MND [23,24]. In ALS patients, repeat AD [21]. Although large repeat expansions do not seem to
length does not seem to affect bulbar or spinal presenta- contribute to the risk of PD, intermediate repeats
tion [47 ,50 ]. between 20 and 39 units have been reported in PD
and essential tremor (ET) patients [15,32,34,35]. Inter-
In non-expansion carriers, no clear association has been mediate repeats were suggested to act as a rare risk factor
identified so far between repeat lengths within the normal rather than a causal factor for PD or parkinsonism since
range and the risk of ALS, FTD or FTD-ALS familial segregation of the intermediate repeat allele with
[17,18,39,48 ,51]. Only one study, a meta-analysis, calcu- clinical PD is lacking and intermediate repeats also occur
lated a significant association of intermediate repeats in controls [15,32] (Figure 2). However, the hypothesis of
between 24 and 30 units with an increased risk of intermediate repeats as a risk factor for PD was not
www.sciencedirect.com Current Opinion in Genetics & Development 2017, 44:117–124
120 Molecular and genetic bases of disease
confirmed in a large study within 7494 clinical PD FTD-ALS patients, but revealed a significant positive
patients [33], nor in autopsy-confirmed PD [52]. Further, correlation between age at onset and expansion size in
intermediate repeats have also been observed in typical both the parietal lobe and cerebellum in ALS patients
PSP patients [25]. only [49 ].
Effect of C9orf72 repeat length on onset age Concerning disease duration, Dols-Icardo et al. could not
and disease duration detect any correlation with repeat size in neither ALS nor
The first hint that repeat length might act as an onset age FTD expansion carriers [47 ], while Suh et al. observed
modifier came from observations of an earlier onset age in that longer repeat expansion sizes were associated with
younger generations of C9orf72 families suggesting a shorter disease durations in individuals who presented
mechanism of anticipation [3–5,11–13,15,16] (Figure 2). with FTD [48 ]. Indirect evidence for an inverse correla-
Not all authors were able to observe this [10,29] and clear tion between repeat expansion size and disease duration
evidence for the occurrence of anticipation was lacking has been provided by the observation of an inverse
until we recently provided genetic and molecular argu- correlation between hypermethylation of the flanking
ments for anticipation [46 ]. We were able to observe an CpG island, which is reported to be associated with repeat
increase in expansion size of about 1000 units from a length [46 ], and disease duration [53,54].
parent to his offspring (Figure 2). Further, a correlation
was found between repeat expansion size (categorized as In brain of FTD and/or MND patients, van Blitterswijk
‘short’ (<80 U) versus ‘long’ (>80 U)) and methylation et al. calculated an association of poorer survival after
0
level of the 5 flanking CpG island, which enabled us to disease onset with cerebellar repeat length greater than
study methylation differences across generations in order 1467 repeat units [44], while Nordin et al. found a corre-
to estimate the occurrence of intergenerational repeat lation between poorer survival and the number of repeats
amplification. A significant increase in methylation level in the parietal lobe [49 ]. No correlation with disease
was apparent in the offspring compared to the parents, characteristics was found for repeat lengths in the spinal
supporting an intergenerational repeat amplification [46 ] cord, muscle, blood, kidney, spleen and skin [49 ].
(Figure 2). As the methylation status of a CpG island is
typically coupled with expression of the associated gene, So far, no significant association could be revealed
this may suggest that the mechanism of anticipation acts between repeat numbers within the normal range and
through C9orf72 gene expression. In vitro C9orf72 expres- age of onset or disease duration of non-expansion FTD,
sion has also been demonstrated to be associated with ALS or FTD-ALS patients [8,40,48 ,50 ,51], AD patients
repeat size [14,46 ]. However, other research groups, who [20] or patients with PD or ET and restless legs [35,36].
measured C9orf72 methylation states only in cases with a
long repeat length found an inverse relationship between
repeat length and methylation degree [53]. Furthermore, C9orf72 repeat expansion sizing
not all C9orf72 families show a decrease in onset age An important remaining issue, in studying repeat length
across generations [3,5,11–13,16] and also a contraction in as a modifying factor, are the difficulties encountered in
number of G4C2 repeats has been observed [46 ]. obtaining exact sizes of the expanded repeat due to
several characteristics of the C9orf72 repeat expansion
While some authors could not reveal a correlation (Figure 3). Further, due to somatic mosaicism, shorter
between onset age and repeat size in blood of neither repeat lengths were observed in blood than in the frontal
ALS nor FTD expansion carriers [47 ,50 ], others cortex [41 ,44], and in the cerebellum shorter and more
observed a (trend toward a) positive correlation between uniform repeat lengths were observed compared to other
age at onset and repeat size [23,41 ,45 ] (Figure 2). How- brain regions and blood [41 ,44,48 ]. Since the expansion
ever, these positive studies used different measures of size in peripheral DNA is shown to correlate with age at
repeat size and some results were possibly due to outlier the time of collection, the shorter expansion size in
patients [45 ]. To our knowledge, we are the only cerebellum relative to peripheral DNA might in part
research group who has provided arguments for an inverse be due to reduced repeat instability in the cerebellum
correlation between onset age and repeat length in blood: [48 ,53]. Gijselinck et al. reported the opposite finding: an
we calculated a significantly higher mean onset age in the expansion of more than 1100 repeat units was identified in
carriers of a repeat expansion between 47 and 80 units the cerebellum of an FTLD patient while the median
than in the carriers of a repeat expansion longer than repeat size in blood as well as frontal and temporal cortex
80 units [46 ] (Figure 2). was only 56 units [46 ]. Also Nordin et al. reported two
patients with a repeat size below 100 units in non-neural
In brain, van Blitterswijk et al. calculated a significant tissues versus 20–40 times larger expansions in the central
positive correlation between age at onset and repeat nerve system (CNS) [49 ]. Nevertheless, it was suggested
length in the frontal cortex of FTD patients [44]. Nordin that the tissue mosaicism would probably not be of
et al. could not confirm this finding in FTD, MND and/or sufficient magnitude that normal C9orf72 repeat lengths
Current Opinion in Genetics & Development 2017, 44:117–124 www.sciencedirect.com
Relationship between C9orf72 repeat size and clinical phenotype Van Mossevelde et al. 121
Figure 3
Difficulties in C9orf72 repeat expansion sizing by Southern blot
1) large quantities of high molecular weight genomic DNA required
2) challenging characteristics of the C9orf72 repeat expansion
100% GC content DNA ladder DNA control expansion carrier 1 expansion carrier 2 expansion carrier 3
∼ massive expansion sizes up to 4400 units 27 kb [23] 23.1 kb highly repetitive flanking sequence 9.4 kb ‘long’ > 388 U 6.6 kb somatic mosaicism and repeat instability
4.4 kb
enormous intra-individual repeat size variability [49] :
repeat size blood ≠ repeat size frontal lobe ≠ repeat size cerebellum... ‘short’ :55-100 U 2.3 kb WT : 2-24 U smears on Southern blot [25] 2.0 kb
Current Opinion in Genetics & Development
Difficulties in C9orf72 repeat expansion sizing by Southern blot.
On the left side, a textbox lists the difficulties for C9orf72 repeat expansion sizing by Southern blot. On the right side, a schematic example of
C9orf72 repeat expansion sizing by Southern blot is shown. For the control individual only a signal in the wild type (WT) region is present, since
both alleles have a length between 2 and 24 units (U). All expansion carriers have one signal in the WT region (for the WT allele) and a second
signal representing the expanded allele. Expansion carrier 1 and 2 carry a ‘long’ repeat expansion, while expansion carrier 3 carries a ‘short’
repeat expansion [46 ].
Abbreviations: kb, kilobase; U, unit; WT, wild type.
would be measured in blood while the CNS would display questions remain unanswered is the technically challeng-
abnormally expanded repeats [55]. ing nature of the repeat sizing, requiring large quantities
of high molecular weight genomic DNA, which is rarely
Conclusions available from affected tissue or even blood. Further
The observed phenotypical heterogeneity in C9orf72 attempts to develop reliable methods that enable accurate
repeat expansion carriers suggests the presence of modi- measuring of large repeat sizes remain essential to unravel
fying factors. Although, in accordance with other repeat the remaining questions regarding the phenotypic modi-
expansion diseases, the C9orf72 repeat expansion size fying effect of the C9orf72 repeat size.
seems the best candidate, conflicting results have been
reported and convincing evidence for a modifying effect Conflict of interest statement
of the repeat size on the clinical phenotype is still lacking. There is no conflict of interest.
Many questions remain unanswered. No clear messages Acknowledgements
can be provided to clinicians concerning diagnostics. Based The authors’ research is in part funded by the Belgian Science Policy Office
Interuniversity Attraction Program, the Flemish Government initiated
on repeat size in blood, one cannot predict if a C9orf72
Impulse Program of Networks for Dementia Research, the Flemish
repeat expansion carrier will develop disease and at what
Government initiated Methusalem Excellence Program, the Research
age, nor which symptoms of the FTD-ALS spectrum will Foundation Flanders and the University of Antwerp Research Fund;
Belgium.
be exhibited. Also, what is the effect and role of somatic
mosaicism in diagnosis heterogeneity? Do longer repeat
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