Transcranial sonography findings in a large family with homozygous and heterozygous PINK1 mutations
- J M Hagenah1,
- B Becker1,
- N Brüggemann1,
- A Djarmati1,2,
- K Lohmann1,2,
- A Sprenger1,
- C Klein1,2,
- G Seidel1
- 1 Department of Neurology, University of Lübeck, Lübeck, Germany
- 2 Department of Human Genetics, University of Lübeck, Lübeck, Germany
- Dr J Hagenah, Department of Neurology, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany;
- Received 11 December 2008
- Revised 2 April 2008
- Accepted 7 April 2008
- Published Online First 9 May 2008
Objective: To investigate substantia nigra (SN) echogenicity in members of a family with homozygous and heterozygous PTEN induced kinase (PINK1) mutations with or without signs of Parkinson’s disease (PD).
Methods: Transcranial sonography (TCS) was used to investigate 20 members of a family with PINK1 mutations, including four homozygous and 11 heterozygous mutation carriers and five individuals with no mutation. For comparison, a healthy control group of 18 subjects without a positive family history of PD (control group) and a healthy control group of 15 subjects with a positive family history of sporadic PD (relative group) were investigated. For statistical analysis, the larger area of the two SNs echogenicity (aSNmax) of each individual was selected.
Results: A significantly increased aSNmax was found for all subgroups compared with the control group. The group of homozygous carriers of a PINK1 mutation had a significantly increased aSNmax compared with all of the other subgroups, except the group of heterozygous mutation carriers.
Conclusions: These findings in carriers of a PINK1 mutation are comparable with those in carriers of Parkin mutations and non-genetic PD. The increased aSNmax in family members without a mutation suggests an additional contributing factor independent of the PINK1 mutation that may also play a role in relatives of patients with sporadic PD.
Mutations in the PTEN induced kinase (PINK1) gene are the second most frequent known cause of early onset parkinsonism after Parkin mutations.1 PINK1 parkinsonism is considered to be inherited in an autosomal recessive fashion.
Clinical signs of parkinsonism as well as a dopaminergic deficit on positron emission tomography have been described in carriers of homozygous and heterozygous PINK1 mutations,1–7 suggesting that a single mutation may be a risk factor for parkinsonism.8 9 Furthermore, there is support on the molecular level for a possible dominant negative effect of heterozygous PINK1 mutations.10
Transcranial sonography (TCS) of the brain parenchyma has previously been used for the detection of an increased area of echogenicity in the substantia nigra (aSN) in homozygous and heterozygous mutation carriers of Parkin mutations compared with controls. This aSN increase was associated with reduced [18F]-dopa uptake in the corresponding putamen11 12 and has been described in both symptomatic and asymptomatic carriers of a heterozygous Parkin mutation. In the present study, we investigated a large family (family W) with four homozygous and 11 heterozygous carriers of a recurrent nonsense mutation in the PINK1 gene (c.1366C>T; p.Q456X)4 by TCS.
PATIENTS AND METHODS
After giving informed consent, 20 family members (family W) with a PINK1 mutation (nonsense mutation; c.1366C>T; p.Q456X), 18 healthy subjects without a family history of PD (“controls”) and 15 first degree relatives of 12 patients with sporadic PD (“relatives”) underwent a detailed neurological examination by a member of the local movement disorders team, including assessment with the Unified Parkinson’s Disease Rating Scale (UPDRS) on and off medication. All family members were videotaped using the UPDRS part III protocol, and all 20 videotapes were evaluated in a blinded random fashion by an independent movement disorder specialist. PD was defined according to the UK Parkinson’s Disease Brain Bank Criteria,13 except that positive family history for PD was not considered an exclusion criterion. Signs of possible and probable PD were rated as previously described.4
The study employed TCS with a SONOS 5500 ultrasound system (Philips Medical Systems, Best, The Netherlands) with a 2.0–2.5 MHz sector transducer (S3 probe). All patients were studied by one experienced ultrasound examiner (GS or JH), who was blinded to the results of the genetic status of the subjects. The examination was performed from both sides and only the ipsilateral SN was evaluated. Images were digitally stored on an optical disc for later offline computer based analysis by an independent investigator (NB), who had not been involved in the TCS examination, using a computer based analysis (Scion Image Beta 4.02 Win software package) by encircling and measuring the area of echogenicity in the SN (aSN). For statistical analysis, the larger aSN of each individual was selected or, in cases of an insufficient bone window on one side, the ipsilateral aSN of the analysable side (aSNmax).
Analysis of variance and subsequent post hoc tests were used to compare groups of mutation carriers, controls and relatives with PD. Rank correlations (Spearman-Rho) were used to test the relationship between echogenicity and clinical scores. Statistics were performed using SPSS V.13.0; the level of significance was set at p<0.05.
Of the 20 family members, four (three women, mean age 66.8 (4.8) years) were homozygous mutation carriers, 11 were heterozygous mutation carriers (two women, mean age 42.4 (5.6) years) and five carried no mutation (three women, mean age 40.6 (10.0) years). Subjects from the group “controls” (seven women and 11 men, mean age 44.6 (9.1) years) were mutation negative. Of the 15 subjects from the group “relatives” (nine women, mean age 43.9 (8.1) years), only two (one woman) were tested directly and in another six cases the affected parent was mutation negative (table 1).
All four homozygous mutation carriers were definitely affected and had clinical signs in both the on and off states. The 11 heterozygous carriers were all unaware of any symptoms (asymptomatic) but six of them displayed slight or mild signs of parkinsonism and were classified as affected.4 Details of the clinical features are described elsewhere.4 None of the control subjects without a family history of PD showed any signs of parkinsonism whereas two young men (both 29 years old) from the “relatives” group with a positive family history of sporadic PD had mild rigidity and bradykinesia of the right arm, of which they were unaware. In one of them, a Parkin or PINK1 mutation was excluded but in the other case, the mutational status could not be obtained.
A sufficient bone window was found on both sides in 79% of the subjects. The mean area of the aSNmax echogenicity was 0.48 (0.12) cm2 in the homozygous mutation carriers, 0.36 (0.12) cm2 in the heterozygous mutation carriers, 0.28 (0.07) cm2 in the family members without mutation, 0.07 (0.05) cm2 in the “controls” group and 0.18 (0.07) cm2 in the “relatives” group (fig 1). No significant difference was found for the aSN in heterozygous mutation carriers with or without signs of parkinsonism. Analysis of variance revealed a significant effect for the factor “group” (controls, relatives, heterozygous mutation or homozygous mutation F(4.48) = 34.48, p<0.001), and post hoc analysis (Bonferroni corrected) revealed significant differences between the “controls” and all PINK1 family member groups, regardless of their clinical or mutational status and also compared with the group of “relatives”. Furthermore, a significant difference was detected between family members without mutations and homozygous mutation carriers (p<0.01). There was no significant difference between the other subgroups of the family. For the “relatives” groups, there was no significant difference in aSNmax compared with the PINK1 family members without a mutation but there was significantly lower echogenicity of the SN compared with the heterozygous and homozygous mutation carriers. No significant correlation was found between the severity of symptoms (UPDRSIII score) and the ipsilateral or contralateral aSN in the possibly, probably and definitely affected individuals. In the three definitely affected (homozygous) family members with a sufficient bone window on both sides, the clinically more affected side was correlated with the size of the contralateral aSN. No such correlation was found in any of the other subgroups.
In this first systematic study of TCS in PINK1 linked parkinsonism, an increased area of aSN was detected in all clinically affected carriers of two (homozygous) mutations, comparable with ultrasound findings in other monogenic forms, such as Parkin and LRRK2 associated parkinsonism.11 12 14 15 The impact of an increased aSN currently remains unknown; possible interpretations include that of a vulnerability marker or a cofactor contributing to the pathogenesis of parkinsonism, irrespective of the aetiology. This finding is, at least in part, comparable but not related to the occurrence of Lewy bodies as a pathological hallmark of idiopathic PD that are found in idiopathic as well as many monogenic forms of the disease.8 16 17 The increased echogenicity is thought to be associated with nigral iron deposition18 and does not seem to serve as a quantitative marker for SN degeneration but rather occurs independently from degeneration of presynaptic dopaminergic nerve terminals.19
The relationship between the clinical, genetic and ultrasound findings is currently unresolved for the most part. Our study expands previous observations of a potential relationship between increased SN echogenicity with a higher number of mutated Parkin alleles11 12 to the PINK1 gene. However, these data need to be interpreted with caution because of the relatively small sample sizes studied. Furthermore, all previously published Parkin family members11 12 and the PINK1 family members from the present study, who carried two mutations, definitely suffered from parkinsonism, whereas only some of the members with a single heterozygous mutation had signs of probable or possible PD.
For the first time, we systematically investigated a subgroup of family members without a mutation that belonged to a family with monogenic parkinsonism. Intriguingly, even healthy members of the PINK1 family, without a mutation, had a significantly increased aSN compared with unrelated healthy controls. This leads to speculations about additional or independent (genetic) factors apart from PINK1 mutations contributing to the increased aSN in this family. For further evaluation we investigated a group of first degree relatives of patients with sporadic PD. Even though we were unable to test all of the subjects in the first degree relative group for mutations in the PINK1 gene, the likelihood of inclusion of a PINK1 mutation carrier in this group is presumably less than 1%. The findings in this group support this notion of an additional, most likely genetic, factor by revealing an increased aSN in 40% of asymptomatic subjects. This result ties in with the only previous TCS study in first degree relatives of patients with sporadic PD that detected an increased aSN in almost half of the relatives.20 Additional PET investigations in subjects from this published study showed that relatives with a larger aSN also had lower 18F-dopa influx constants (Ki) of the putamen than relatives with a smaller aSN. These findings suggest an additional, as yet unknown, genetic contribution to the PD phenotype in the “sporadic” patients. Further evidence for an as yet unknown genetic factor for increased aSN was reported in a recently published large cross sectional study: the most significant factor associated with SN hyperechogenicity was a family history of parkinsonism.21
Competing interests: None.
Ethics approval: Ethics approval was obtained.
Patient consent: Obtained.