Clinical findings and genetic screening for copy number variation mutations in a cohort of South African patients with Parkinson's disease.
Studies from Africa that have looked at an association between PD and gene mutations are limited, and this report describes the clinical findings of a group of black SA patients with PD who were screened for copy number variation (CNV) mutations in the known PD-causing genes. Whole-exon deletions or duplications, primarily in the parkin gene, are a common cause of PD and have been found in both familial and sporadic forms of the disorder. 
All black African patients with a diagnosis of PD who were being followed up at the neurology clinic at Steve Biko Academic Hospital, Pretoria, SA, were included in the study. The patients were invited to participate in the study, and informed consent and ethical committee approval of the University of Pretoria (Ref. No. 128/2012) were obtained. The patients were evaluated by a clinician proficient in the neurological examination of patients with PD to confirm that they met the UK Parkinson's Disease Society Brain Bank clinical diagnostic criteria for PD. History and demographic data were obtained, and after a full neurological examination, symptoms were scored according to the Unified Parkinson's Disease Rating Scale (UPDRS). Atypical features were noted and patients were classified according to age at onset, cognitive functioning and motor features.
Venous blood (20 mL) was obtained and genomic DNA was extracted. The multiplex ligation-dependent probe amplification (MLPA) method was used for detection of CNV mutations. Specifically, the commercially available SALSA P051-D1 Parkinson kit (MRC-Holland, Netherlands) was used. This kit contains probes for all exons of genes in which PD-causing CNV mutations have previously been found, including parkin, PINK1, DJ-1 and SNCA. In addition, the kit contains probes for detection of two point mutations, G2019S in LRRK2 and A30P in SNCA. The MLPA experiments were performed according to the manufacturer's instructions and the amplified products were analysed on a 3130 x 1 Genetic Analyser (Applied Biosystems, USA). Coffalyser.Net software (vl4072.1958, MRC-Holland, Netherlands) was used for copy number calculations.
Sixteen black patients with PD were identified (9 males and 7 females), 2 of them siblings. Most patients were Sotho speakers (9/16), the rest speaking Zulu or Ndebele. Their ages ranged from 56 to 82 years (mean 68), disease duration from 1 to 21 years (mean 6.8) and age of onset of the disorder from 50 to 74 years; a family history of PD was obtained in 3 patients (19%), the 2 siblings and 1 other patient. Classification according to motor features showed 44% to be mixed, 31% akinetic-rigid and 25% tremor-dominant subtypes. UPDRS scores when on medication ranged from 7 to 88; tremor graded according to the UPDRS was scored as 1 (slight and infrequent) in 4 participants (25%), 2 (mild and impersistent) in 6 (38%), 3 (moderate and mostly present) in 4 (25%) and 4 (marked and mostly present) in 2 (13%). The rigidity score was 1 in 25%, 2 in 50% and 3 in 25%, and the bradykinesia score was 1 in 44%, 2 in 25%, 3 in 13% and 4 in 19%. Cognitive screening using the minimental state examination (MMSE) showed normal cognition in 40% and minimal cognitive impairment in 40%, while 20% had dementia. Patients with cognitive impairment showed a greater mean rigidity score (2.33) as derived from the UPDRS than patients without dementia (1.5). Interestingly, hyperreflexia was found in 7/16 patients (44%), and slowing of saccadic eye movements was seen in 2. The clinical findings are set out in Table 1.
Genetic screening was performed on 11 unrelated patients for whom DNA was available. Importantly, both the G2019S LRRK2 and A30P SNCA mutations were excluded as a cause of the disorder in all the patients. In 10 patients, no CNV mutations were found; however, in one patient a heterozygous deletion of parkin exon 4 was identified. This patient (Table 1, patient 4) had an age of onset of 50 years with no family history of the disorder. In addition to the signs of PD, she was also noted to have hyperreflexia with bilateral Hoffmann's reflexes, but normal plantar responses. No dystonia was noted, and a computed tomography scan of the brain did not show any focal lesions.
PD is one of the commonly occurring neurodegenerative disorders, yet little information about this disease in black African patients is available and clinical and genetic studies involving these patients are limited.
It is well known that PD is mainly seen in older individuals, with the incidence rising with every decade of age; worldwide, ~4-10% of patients have early-onset disease.  Compared with patients with PD from Zambia, in whom the mean age of onset was 54 years,  and a group of 18 black SA patients with PD with a mean age of onset of 52 years,  our patients' mean age of onset at 62.5 years was slightly older; it is, however, comparable to the average age of onset of 62 years in Western studies. There were no patients with young-onset (defined as <50 years) PD in our group, but this may be a reflection of the small sample size.
Clinical classifications of motor subtypes in PD in Western population studies show a mixed subtype of disease as the most common presentation (66%), followed by the akinetic-rigid subtype (26%) and lastly the tremor-dominant group (8%).  Our patients showed a similar pattern, but the tremor-dominant group was larger at 25%. This is comparable to a group of Nigerian patients with PD, 55% of whom had mixed disease, 32% tremor-dominant disease and 14% the akinetic-rigid form.  Eventually this may have prognostic implications, as some studies have shown that patients with tremordominant disease may have a more favourable course. 
Cognitive impairment and dementia may occur in patients with PD, especially after long disease duration. Generally, patients with PD have an almost six-fold increased risk of developing dementia, and up to 24-31% have cognitive impairment.  Of our group of patients, 20% had dementia; these patients all also had high UPDRS scores (>35). Severe motor disorders such as rigidity and gait instability have been linked to an increased probability of developing dementia, and this appeared to be the case in our patients, among whom the mean rigidity score was high in those who were cognitively impaired (2.33 v. 1.5 in non-demented patients), but larger numbers of patients need to be studied to confirm this association. An older age at disease onset was not found to be a risk factor for dementia in our study (ages 50, 60 and 51 years at disease onset in the patients with dementia). Disease duration, age of onset and motor symptom severity have been found to be risk factors in some studies, but others have not confirmed these associations. 
There is currently increasing evidence that genetic factors are important in the development of PD, and up to 10% of cases are believed to have a genetic cause. Genes associated with an autosomal dominant pattern of inheritance include SNCA and LRRK2, while autosomal recessively inherited gene abnormalities include parkin, DJ-1, PINK1 and ATP13A2.  Whereas SNCA and parkin mutations are usually associated with early-onset PD, LRRK2 mutations, specifically the G2019S mutation  (which was not found in our study), are the most frequent cause of late-onset familial and sporadic PD. A positive family history was found in up to 2.4% of black African patients in case series,  and one SA study of a mixed group of patients found an overall positive family history in approximately one-third of individuals with late-onset PD. 
Although the number of patients in our study was small, three reported a positive family history, and only one of the patients (who had reported no family history) had a pathogenic CNV mutation in the parkin gene. As parkin is associated with an autosomal recessive form of PD, further studies are warranted to possibly identify a second pathogenic mutation in this patient.
To date, only two black SA patients have been shown to harbour mutations in the parkin gene and none have had mutations in any of the other PD-causing genes. [17,18] These findings suggest that unravelling the genetics of PD is still only in the early stages, and further studies for new candidate genes are needed. Future studies will involve whole-exome sequencing of DNA from our patients to identify defects in potentially novel PD-causing genes.
Acknowledgments. We thank Genevie Borrageiro for performing the MLPA experiments and Prof. E Janse van Rensburg from the Department of Human Genetics, University of Pretoria, for extraction of the patients' DNA.
Conflicts of interest. None. SB has been awarded grants from the South African Medical Research Council and the National Research Foundation.
Author contributions. C-MS and ACM did the initial planning, conduct and reporting and writing up of the work, SB was in charge of the genetic part of the work and helped with the final drafting of the article, and JAC was involved in the final writing up and editing of the article. All the authors saw and approved the final article.
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Accepted 23 February 2016.
A C Mahne, (1) MB ChB, MMed (Neurol); J A Carr, (2) MB ChB, PhD; S Bardien, (3) PhD; C-M Schutte, (1) MB ChB, MMed (Neurol), MD
(1) Department of Neurology, School of Medicine, Faculty of Health Sciences, University of Pretoria, South Africa
(2) Division of Neurology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, Cape Town, South Africa
(3) Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, Cape Town, South Africa
Corresponding author: C-M Schutte (firstname.lastname@example.org, email@example.com)
Table 1. Clinical features of patients with PD Patient No. 1 2 3 4 5 Sex M F M F F Age (years) 64 82 73 56 63 Age of onset 61 63 68 50 58 (years) Duration (years) 3 21 5 6 5 Family history No No No No No History of falls No No Yes No No Other medical HT, BPH OA HT, No HT conditions heart failure Hyperreflexia Yes No No Yes No Slow saccades No No No No No MMSE 29/34 38 33 24/30 37 UPDRS score 7 25 34 36 9 Genetic Negative Negative Not Heterozygous Not screening done exon 4 done (copy number parkin variation; deletion G2019S in LRRK2; A30P in SNCA) 6 7 8 9 10 11 Sex M M F M F M Age (years) 58 81 76 64 75 62 Age of onset 51 66 74 63 69 60 (years) Duration (years) 7 15 2 1 6 2 Family history No No Yes No No No History of falls No No No No No No Other medical BPH HT HT HT, DM HT No conditions Hyperreflexia Yes No Yes Yes Yes No Slow saccades Yes No No No No No MMSE 8/34 25/34 34/34 Normal 24/30 16/30 UPDRS score 52 70 24 25 69 39 Genetic Not Not Nega- Nega- Nega- Negative screening done done tive tive tive (copy number variation; G2019S in LRRK2; A30P in SNCA) 12 13 14 15 16 Sex M F M M F Age (years) 79 58 64 65 74 Age of onset 69 50 60 57 67 (years) Duration (years) 10 8 4 8 7 Family history No Yes No Yes No History of falls No Wheel- Yes No Yes chair bound Other medical HT, DM, HT HT HT, DM HT, conditions hypothy- cervical roidism CA Hyperreflexia No No Yes No No Slow saccades No No No Yes Yes MMSE 20/24 Severe 25/30 35 18/23 dementia UPDRS score 41 88 34 32 84 Genetic Negative Negative Nega- Sibling Negative screening tive of (copy number patient variation; 8 G2019S in LRRK2; A30P in SNCA) M = male; F = female; HT = hypertension; BPH = benign prostatic hypertrophy; OA = osteoarthritis; DM = diabetes mellitus; CA = carcinoma.
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|Author:||Mahne, A.C.; Carr, J.A.; Bardien, S.; Schutte, C.-M.|
|Publication:||South African Medical Journal|
|Article Type:||Clinical report|
|Date:||Jun 1, 2016|
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