Parkinson’s disease (PD; MIM #168601) is a common neurodegenerative disorder with a prevalence of >1% in populations over 60 years of age (1). PD is clinically characterized by rigidity, bradykinesia, tremor, and postural instability, and may be accompanied with dementia and depression (2, 3). The disease etiology is likely to be multifactorial, involving complex interactions between genetic and environmental factors. In the past 20 years, genetic studies of PD families have provided strong support for the hypothesis that PD has a significant genetic component. To date, 13 genes have been described for hereditary PD (4, 5), and at least 10 of these genes are associated with autosomal recessive (AR) forms of PD. Although mutations in PARK2 (PRKN; MIM #602544), PARK6 (PINK1; MIM #605909), and PARK7 (DJ1, MIM #606324) are infrequent in the PD population, they are responsible for a majority of early-onset PD and are usually known to cause typical PD with indistinguishable clinical signs (1, 4, 6). Moreover, the other, more rare mutations, including PARK9 (ATP13A2; MIM #610513), PARK14 (PLA2G6; MIM #612953), PARK15 (FBXO7; MIM #260300), PARK19 (DNAJC6; MIM #615528), PARK20 (SYNJ1; MIM #615530), PARK23 (VPS13C; MIM #616840), and PODXL (MIM #602632) have been implicated in parkinsonism characterized by juvenile onset and atypical clinical signs (5, 7–10).

PRKN, located on chromosome 6q26, is one of the largest human genes spanning 1.38 Mb with 12 exons and large intronic regions (11). Mutations in this gene explain up to 50% of AR PD and about 15% of sporadic cases with early onset (12, 13). A large number of mutations have been identified in all populations studied, regardless of ethnic origin, including exon rearrangements and point mutations. PINK1 contains eight exons that span 1.8 kb and encode a 581-amino-acid serine/threonine kinase protein. This protein exhibits an N-terminal mitochondrial targeting sequence, a putative transmembrane anchor as well as a C-terminal kinase domain (14, 15). It was noticed that the mutational hotspot is found in exon 7. More than 70 mutations have been reported in PINK1; two-thirds of which are loss-of-function mutations affecting its kinase domain (16). DJ1 is composed of eight exons spanning 24 kb and encodes a 189-amino-acid protein (17, 18). The causative PD mutations identified in DJ1 are mainly point and structural mutations resulting in loss of protein function (17, 19). All PRKN, PINK1, and DJ1 mutation carriers present with similar clinical features that are practically indistinguishable from idiopathic forms of PD, including a good response to levodopa with a tendency to develop levodopa-induced dyskinesia and a slow progression.

In addition, mutations in ATP13A2 cause a juvenile parkinsonism characterized by a rapid progression, supranuclear gaze palsy, pyramidal signs, and dementia (4, 20). Mutations in PLA2G6 display both early-onset and juvenile forms of recessively inherited atypical parkinsonism. The early-onset form is associated with levodopa-responsive dystonia-parkinsonism, pyramidal signs, cognitive dysfunction, and Lewy body disease accumulation in the brain, whereas the juvenile one is distinguished by dystonia, cerebellar ataxia, spasticity in all limbs, and cognitive decline (21). Mutations in FBXO7 are also responsible for a juvenile form that presents with early dystonia and pyramidal signs (22). Furthermore, the gene DNAJC6 encodes for the HSP40 Auxilin protein. Mutations in DNAJC6 induce an early-onset parkinsonian-pyramidal phenotype with rapid progression and a poor response to levodopa. Patients with these mutations can also show juvenile parkinsonism with a relatively slow disease progression (7, 23, 24). Also responsible for early-onset PD, SYNJ1 mutations cause atypical parkinsonism with a number of features, such as dystonia, oculomotor apraxia, dementia, and seizures (25–27). VPS13C is a multi-exonic gene (86 exons) responsible for early-onset parkinsonism, and it is associated with a severe phenotype characterized by rapid progression, cognitive deterioration, and a widely distributed presence of Lewy bodies (9, 23). Recently, a homozygous frameshift mutation in the PODXL gene was described as a causal factor for juvenile parkinsonism. Although these data are currently insufficient to support this assertion, the clinical features appear similar to those of classic PD (10).

In the present study, we analyzed the genetic bases of a series of consanguineous PD patients from Morocco by combining chromosomal microarray analysis (CMA) and gene panel next-generation sequencing (NGS) to target 22 genes associated with PD and overlapping phenotypes.

Of the 145 PD patients recruited during the 4-year period, 19 individuals were born from a consanguineous marriage, which represents an inbreeding rate of 13%. Of them, seven patients were males (37%) and four had a positive family history of PD (21%). The mean age at examination was 56.2 years (range 19–84) and the mean age at onset was 47.4 (range 12–77) years. Sanger screening for the LRRK2 exon 41 showed the G2019S mutation in the homozygous state for one patient without family history of the disease (Table 1, patient ID 3332). The clinical phenotype of this patient, whose disease onset was recorded at 48 years and who had a disease duration of 9 years, is no different from the heterozygous G2019S carriers described previously (29). Because the G2019S mutation is very common in Morocco, this patient will be included in a large series of G2019S carriers to be analyzed for the dopamine metabolism genes in order to determine their effects on age of onset, clinical phenotype and response to treatment.

The remaining 18 patients negative for the LRRK2 G2019S mutation were subjected to high-resolution CMA using an Affymterix platform and CytoscanHD microarrays, which revealed microdeletion chromosomal region 6q26 of the PRKN in 3 of the 18 consanguineous PD patients (16.7%). The deletions were determined to be homozygous in two patients and compound heterozygous in one patient. Data collected from Patient 3020, who carries a compound heterozygous deletion, are shown in Figure 1. This male patient (III.3) is a sibling to four other, unaffected children born from a common consanguineous marriage of the first degree (Figure 1A). Despite the patient’s consanguineous heritage, the allele-difference plot did not show ROH at chromosome 6q (Figure 1B). The weighted log₂ ratio and copy-number-state plots of chromosomal region 6q26 show two heterozygous PRKN deletions of 166 and 587 kb encompassing exons 3–4 and exons 3–7, respectively (Figure 1C). The centromeric and telomeric break points of the smallest deletion were located at positions 162.600.881 and 162.767.227, and the break points of the largest deletion were located at positions 162.195.309 and 162.783.147, respectively. Absence of ROH at region 6q26 suggests that the two deletions were inherited from two different ancestors, and thus the patient’s father (II.1) and mother (II.2) must carry one of the two mutations in the heterozygous state.

In addition, patient 3158 (III.6), a 49-year-old male, was also born from a consanguineous marriage of the first degree (Figure 2A). The allele-difference plot shows a 6.5 Mb ROH at chromosome 6q (Figure 2B). The weighted log₂ ratio and copy-number-state plots of the 6q26 region show a homozygous deletion of 154 kb that encompasses exon 9 of the PRKN gene (Figure 2C). The centromeric break point of the deletion was located within intron 9 (161.835.769), and the telomeric break point was located in intron 7 (161.990.516).

Data collected from patient 3468, who is also homozygous for a PRKN gene deletion, are shown in Figure 3. This patient (III.1) was born from a first-degree consanguineous marriage and has a brother known to have PD (Figure 3A). The allele-difference plot shows three long, contiguous stretches of homozygosity at chromosome 6q: (1) 12.49 Mb from 6q16.1-q21, (2) 15.79 Mb from 6q22.31-q23.3, and (3) 17.2 Mb from 6q25.2-qter (Figure 3B). The weighted log₂ ratio and copy-number-state plots of the 6q26 region show a homozygous deletion of 339kb encompassing exons 6 and 7 of the PRKN gene (Figure 3C). While the centromeric break point of the deletion was located within intron 7 (162.101.291), the telomeric break point was located in intron 5 (162.441.087).

While Patients 3158 and 3468 presented with early-onset PD with mixed phenotypes, patient 3020 presented with juvenile-onset disease with a phenotype of tremors and dystonia. The three patients were improved by very low doses of antiparkinsonian drugs without motor fluctuations or dyskinesia, even after up to 35 years of disease duration in Patient 3020 (Table 1).

For patients without PRKN exon deletions, the high-resolution karyotype was normal and no CNV was identified within the known PD loci. However, because Patients 3528 and 3223 showed ROH covering the 1p36.12 locus, they were also sequenced for the candidate PINK1 gene. Results revealed two homozygous point mutations: Q456X (c.1366C > T) (Figures 4A,B) mutation in exon 7 and novel L539F (c.1617G > C) variation in exon 8 (Figures 4C,D). The patient with the Q456X mutation showed an early-onset disease, mixed phenotype without balance impairment after 20 years of disease duration, dyskinesia with very low dose levodopa therapy (400 mg/day), and a mild degree of cognitive impairment. Interestingly, the patient with the L539F mutation manifested parkinsonism at 50 years of age with high doses of levodopa (1000mg/day) after 4 years of disease progression (Table 1).

Of the PRKN mutations reported so far, large deletions are the most frequent and can be present in either homozygous or compound heterozygous states (16, 33, 34). However, due to limitations of the multiplex ligation-dependent probe amplification (MLPA) and quantitative PCR methods used commonly, only few studies have determined the size and break point locations of the rearrangements. We overcame this issue by employing high-resolution CMA, which allows for rapid and effective detection of CNV and their break point locations. Through use of Cytoscan HD arrays, the screening of 18 consanguineous, Moroccan PD patients revealed four deletions in three probands. Our results show a rearrangement frequency of 16.7% in consanguineous Moroccan PD patients. The deletions were determined to be homozygous in two patients and compound heterozygous in one patient. All the rearrangements observed in our sample were deletions located between exons 2 and 9, and their break point locations appear to be unique. In patient 3020, although the two deletions were inherited from two individuals of the same, highly consanguineous family, the break point locations of their intron were different. These findings suggest that the deletions were independent and recurrent events, which confirms previous studies reporting that most PRKN gene deletions occurred between exons 2 and 8. This region of PRKN comprises the FRA6E center and is considered a deletion hotspot because it contains certain microhomology sequences that have been frequently implicated in the main rearrangement process (33, 35, 36).

Moreover, in addition to the detection of CNV, the CMA method enables detection of ROH, which is effective for identifying candidate genes of recessive diseases. Indeed, two patients without PRKN CNV showed ROH at the 1p36 locus and had mutations in the PINK1 gene. These mutations consisted of the already known Q456X mutation in exon 7 and a new L539F variant in exon 8; the latter of which is located in the C-terminal domain of the PINK1 protein that we have already shown to be probably pathogenic (37).

Furthermore, we demonstrate the feasibility of NGS as a research and potential diagnostic tool for patients with parkinsonism. For instance, we identified the same homozygous nonsense mutation of p.W258X in ATP13A2 in two unrelated consanguineous patients, probably resulting from a common founder. This mutation falls into an ROH at chromosome 1p, detected in one patient. We also found two different heterozygous variants in SYNJ1 in one patient: a novel frameshift mutation and a missense change that is likely benign.

Clinically, our patients with mutations in PRKN and PINK1 presented with a classical PD phenotype with early onset, good response to levodopa, and benign course as previously reported in the literature (12). It should be noted that the patient sharing two compound heterozygous deletions in PRKN with a juvenile onset, tremoric phenotype, and dystonia, did not exhibit non-motor symptoms after 35 years of disease duration. Also, for the patient with the PINK1 stop mutation, postural instability was not seen after 20 years of disease duration. Otherwise, mutations in ATP13A2 was reported to cause Kufor–Rakeb syndrome with juvenile onset and atypical clinical features including pyramidal signs, dystonia, cognitive decline, supranuclear gaze palsy (38) and may become unresponsive to levodopa as the disease progresses (39). Our patients who had the same novel R258X mutation had few atypical signs, namely dystonia and cognitive impairment, but one patient also displayed bulbar symptoms at the onset of the disease. These bulbar symptoms can be explained by either bilateral pyramidal signs (pseudo-bulbar syndrome). Furthermore, mutations in SYNJ1 have been reported previously to cause AR PD with symptom onset occurring after about 30 years of age and with disease progression ranging from severe to stable. Also, it has been observed that SYNJ1 mutations induce severe dyskinesia with generalized seizures in childhood, dystonia, developmental delay, cognitive impairment, and oculomotor disturbances even when coupled with low-dose levodopa therapy (25, 40). However, we report here a male patient heterozygous for a novel frameshift mutation in SYNJ1 who displays a late-onset, slowly progressive form of typical PD and responds well to low-dose levodopa therapy without apparent cognitive impairment. Though unavailable for examination, his father also had parkinsonism, who by the patient’s accounts followed a similarly benign course. This case is thus consistent with a pseudo-dominant pattern of inheritance or recessive inheritance in compound association with another heterozygous mutation in a gene that not exists in the panel used.

In conclusion, this study provides evidence that supports the power and efficiency of the high-resolution CMA method for uncovering micro-rearrangements in the PRKN gene for patients with AR PD. By focusing on patient populations with a high prevalence of consanguinity in Morocco, we also demonstrate the potential of this approach to be used a first-line diagnostic test. Furthermore, the combination of CMA with NGS provides an important understanding of the genetic bases of AR PD.

This study was approved by the biomedical research ethics committee of the Medical School of Rabat (CERB) and written informed consent was obtained from all subjects in accordance with the Declaration of Helsinki.

WR, HT, MR, and ZS phenotyped patients. RB and NB performed DNA extraction and banking. RB performed Sanger Sequencing. ABo performed microarray analysis in the Medical School of Rabat. CT and VD performed NGS analyses at the ICM platform. SL and AB supervised the work at the ICM institute. ABo, ABe, and MY supervised the work and obtained funding support. ABo and RB wrote the manuscript. All authors edited the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.