We screened a group of 567 dystonia patients and identified 10 novel variants (1.8%), including six missense (1.1%) and a frameshift variant (0.2%). Based on functional analysis, we consider six of them (Arg13His, Lys16Glu, His23Pro, Lys24Glu, Pro26Leu, Asp191Thrfs*
9) to represent mutations, ie to be pathogenic (1.1%). The detected non-coding variants are unlikely to be pathogenic as they do not affect the protein sequence. However, it is conceivable that they alter the expression efficiency but proof is lacking due to unavailability of the respective biological material such as RNA or affected family members to test for segregation. The missense variant Ile80Val seems to represent a benign alteration as indicated by the remaining full THAP1 activity in the in-vitro
assay and as also calculated by both prediction tools. This finding is supported by three additional notions: first, the amino acids isoleucine and valine have a similar structure and both belong to the unpolar amino acids. Second, Campagne et al21
revealed lysine 70 as the most C-terminal amino acid residue responsible for DNA binding by detailed structural determination of the THAP domain. Thus, isoleucine 80 should not be involved in DNA binding. Third, the patient has a rather late age at onset (41 years) and speech is not affected (see below). On the other hand, lack of an effect in the reporter gene assay might be specific to the tested target promoter or, alternatively, isoleucine 80 may be involved in another function of THAP1, such as protein–protein interactions.
THAP1 contains a bipartite NLS spanning 16 amino acids (aa 146–162) in the C-terminal part of THAP1.18
Although the frame-shift mutation Asp191Thrfs*
9 does not affect the NLS itself, we demonstrated an impaired nuclear import of mutant THAP1 in vitro
. This altered intracellular distribution of THAP1 may be explained by a protein misfolding affecting at least the C-terminal region of THAP1, which disturbs the formation of the bipartite NLS and results in a reduced interaction with nuclear importing proteins. Alternatively, it is also conceivable that there is remaining THAP1 nuclear import and function in vivo
and the variant is benign. The DYT6-atypical phenotype in our patient with late onset (49 years) and focal dystonia without involving speech actually supports this possibility.
The results of prediction programs for functional effects of the mutations and the effect size measured in vitro clearly correspond for four of the six missense changes including the Ile80Val variant. For Arg13His, however, an effect on function was predicted by both programs but experimental evidence suggests a rather mild effect. The results for the Lys24Glu mutation were more conflicting as experimental evidence indicates a medium effect but one of the prediction programs (PolyPhen2) predicted no functional change at all.
We detected a THAP1 mutation in 1.1% of our mainly focal dystonia patients. Mutation frequency in the literature ranges from about 0.5–1.8% for mainly unselected primary dystonia patients.9, 10, 11, 12, 13, 15
mutations are rare in dystonia patients. About 50 different THAP1
mutations have been reported to date.2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 22
One-third of the mutations represents missense mutations located in the THAP domain and is thought to interrupt DNA binding. Another one-third of the mutations are considered to disturb the NLS, such as nonsense mutations, small insertions/deletions, or missense mutations within the NLS. For these proteins, the nuclear import is disturbed resulting in impaired transcriptional activity. Thus, in about 70% of the reported mutations, the DNA binding of mutant THAP1 is considered to be affected. However, functional proof for most of the reported mutations is missing. In the present study, we show that the rare missense variant Ile80Val is probably not pathogenic.
Regarding, the non-coding variants c.-237_236delinsTT, we confirm that there is no significant association with dystonia. Interestingly, however, most studies demonstrate a higher (but not significantly higher) frequency of the variant in dystonia patients compared with controls.9, 10, 23, 24
The association may occur only in subtypes of dystonia. To date, the total number of carriers is too small to obtain significant differences for this rare variant. A differential effect in dystonia subtypes may also be the explanation for the lack of any trend in another study.11
In contrast to our study design, these authors included only about 20% patients with focal dystonia and 75% of the patients had an age of onset <30 years.11
These inclusion criteria are likely to preferentially select patients with monogenic causes of dystonia rather than those carrying polygenic risk factors, thereby masking a possible association. The c.71+9C>A was too rare in our sample to evaluate any trend of a possible association with dystonia.
Among the about 100 described mutation carriers, most presented with an early onset and cervical or arm dystonia at onset.6, 7, 8, 9, 10, 11, 12, 13, 14, 15
In more than 80% of the patients dystonia spreads to other body parts. Speech was affected in about 70% of patients. Clinical phenomenology in mutation carriers reported here was similar. All had onset of symptoms in the neck or arm. All but one mutation carrier had an age at onset between 6 and 11 years and presented with generalized (n
=3) or segmental (n
=2) dystonia. Only the carrier of the frameshift mutation close to the end of THAP1 had a late onset at the age of 49 years and dystonia remained focal for 10 years after onset. It can be speculated that the effect of this mutation may be rather mild compared with the missense mutations as a considerable proportion of the protein can still enter the nucleus. We also verified the high frequency of laryngeal involvement (4/6) ranging from mild spasmodic dysphonia to aphonia.
Finally, we aimed to correlate the severity of missense changes in the DNA-binding domain with clinical features. Interestingly, the carriers of the benign Ile80Val variant had a rather mild, DYT6-atypical phenotype with a late age at onset, focal dystonia, normal speech, and a negative family history. Further, the carriers of the two mutations with the highest effect on protein function were the only two with a positive family history (), suggesting that these mutations may exhibit a higher penetrance. To date, these correlations are highly speculative and investigations of larger samples will reveal if in-vitro functional analyses can be correlated to the phenotypic presentation.
Taken together, we report 10 novel variants in THAP1 and demonstrate a functional effect for six of them, underlining that functional analysis is necessary to distinguish between benign variants and pathogenic mutations.